DNA encoding SNORF25 receptor

ABSTRACT

This invention provides isolated nucleic acids encoding mammalian SNORF25 receptors, purified mammalian SNORF25 receptors, vectors comprising nucleic acid encoding mammalian SNORF25 receptors, cells comprising such vectors, antibodies directed to mammalian SNORF25 receptors, nucleic acid probes useful for detecting nucleic acid encoding mammalian SNORF25 receptors, antisense oligonucleotides complementary to unique sequences of nucleic acid encoding mammalian SNORF25 receptors, transgenic, nonhuman animals which express DNA encoding normal or mutant mammalian SNORF25 receptors, methods of isolating mammalian SNORF25 receptors, methods of treating an abnormality that is linked to the activity of the mammalian SNORF25 receptors, as well as methods of determining binding of compounds to mammalian SNORF25 receptors, methods of identifying agonists and antagonists of SNORF25 receptors, and agonists and antagonists so identified.

This application is a continuation-in-part of U.S. application Ser. No.09/255,376, filed Feb. 22, 1999, the contents of which are herebyincorporated by reference into the subject application.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to bypartial citations within parentheses. Full citations for thesepublications may be found at the end of the specification immediatelypreceding the claims. The disclosures of these publications, in theirentireties, are hereby incorporated by reference into this applicationin order to more fully describe the state of the art to which theinvention pertains.

Neuroregulators comprise a diverse group of natural products thatsubverse or modulate communication in the nervous system. They include,but are not limited to, neuropeptides, amino acids, biogenic amines,lipids, and lipid metabolites, and other metabolic byproducts. Many ofthese neuroregulator substances interact with specific cell surfacereceptors, which transduce signals from the outside to the inside of thecell. G-protein coupled receptors (GPCRs) represent a major class ofcell surface receptors with which many neurotransmitters interact tomediate their effects. GPCRs are characterized by sevenmembrane-spanning domains and are coupled to their effectors viaG-proteins linking receptor activation with intracellular biochemicalsequelae such as stimulation of adenylyl cyclase.

Vitamin A₁ (all-trans-retinol) is oxidized to vitamin A₁ aldehyde(all-trans-retinal) by an alcohol dehydrogenase. All-trans-retinal iscritical for the synthesis of rhodopsin in retinal cells, where it playsa key role in the visual system. All-trans-retinal can also be convertedto all-trans-retinoic acid (ATRA) by aldehyde dehydrogenase and oxidasein other cell types (Bowman, W. C. and Rand, M. J., 1980).

Historically, ATRA and the other active metabolites of vitamin A,9-cis-retinoic acid (9CRA), were thought to only mediate their cellulareffects through the action of nuclear retinoic acid receptors (RARα, β,γ) and retinoid X receptors (RXRα, β, γ) (Mangelsdorf, D. J., etal,1994). These receptors are members of a superfamily ofligand-dependent transcription factors, which include the vitamin Dreceptor (VDR), thyroid hormone receptor (TR), and peroxisomeproliferator activator receptors (PPAR). They form heterodimers andhomodimers that bind to DNA response elements in the absence of ligand.In response to ligand binding the dimer changes conformation which leadsto transactivation and regulation of transcription of a set(s) of celltype-specific genes (Mangelsdorf, D. J., et al,1994; Hofman, C. andEichele, G., 1994; and Gudas, L. J. et al, 1994).

Since retinoic acid produces a wide variety of biological effects, it isnot surprising that it is proposed to play an important role in variousphysiological and pathophysiological processes. Retinoids controlcritical physiological events including cell growth, differentiation,reproduction, metabolism, and hematopoiesis in a wide variety oftissues. At a cellular level, retinoids are capable of inhibiting cellproliferation, inducing differentiation, and inducing apoptosis(Breitman, T. et al, 1980; Sporn, M. and Roberts, A., 1984, and Martin,S., et al, 1990). These diverse effects of retinoid treatment prompted aseries of investigations evaluating retinoids for cancer chemotherapy aswell as cancer chemoprevention. Clinically, retinoids are used for thetreatment of a wide variety of malignant diseases including: acutepromyelocytic leukemia (APL), cutaneous T-cell malignancies,dermatological malignancies, squamous cell carcinomas of skin and of thecervix and neuroblastomas (Redfern, C. P. et al, 1995 for review).Retinoids have also been examined for their ability to suppresscarcinogenesis and prevent development of invasive cancer. 13-cisretinoic acid reverses oral leukoplakia, the most common premalignantlesion of the aerodigestive tract, and is also used in thechemoprevention of bladder cancer (Sabichi, A. L. et al, 1998, forreview). Also, 13-cis retinoic acid treatment as adjuvant therapy aftersurgery and radiation in head and neck cancer caused a significant delayin the occurrence of second primary cancers (Gottardis, M. M. et al,1996, for review).

Interestingly, retinoids also have an effect on pancreatic function. Ithas been demonstrated that retinoic acid (or retinol) is required forinsulin secretion from isolated islets (Chertow, B. S., et al, 1987) andfrom RINm5F rat insulinoma cells (Chertow, B. S., et al, 1989). Retinoicacid may also have an effect on cell-to-cell adhesion and aggregation(Chertow, B. S., et al, 1983). In addition, a single intragastricadministration of 9CRA (but not ATRA) induced a wave of DNA synthesis inthe pancreatic acinar cells and in the proximal tubular epithelial cellsof the kidneys (Ohmura, T., et al, 1997). Therefore, retinoic acid couldplay a role in the normal pancreatic function and possibly in thedevelopment of diabetes. There is also some evidence that retinoidscould be useful in the treatment of pancreatic malignancies(El-Metwally, T. H. et al, 1999; Rosenwicz, S. et al, 1997; andRosenwicz, S. et al, 1995).

Retinoids have been shown to affect epidermal cell growth anddifferentiation as well as sebaceous gland activity and exhibitimmunomodulatory and anti-inflammatory properties. Therefore, retinoidshave been increasingly used for treatment of a variety of skin disordersincluding: psoriasis and other hyperkeratotic and parakeratotic skindisorders, keratotic genodermatosis, severe acne and acne-relateddermatoses, and also for therapy and/or chemoprevention of skin cancerand other neoplasia (Orfanos, C. E., et al, 1997 for review).

Retinoids are also involved in lung development. Fetal lung branchingleading to development of the alveolar tree is accelerated by retinoicacid. Currently, prematurely delivered infants who have immature lungsare treated with vitamin A, but other applications may exist thatrequire further investigation (Chytil, F., 1996).

Lastly, there is some evidence that suggests that retinoids may play arole in schizophrenia (Goodman, A. B. 1998) and Alzheimer's disease(Connor, M. J. and Sidell, N., 1997).

The extensive list of retinoid-mediated effects indicate that retinoicacid receptors (non-nuclear) are attractive as targets for therapeuticintervention for several disorders and would be useful in developingdrugs with higher specificity and fewer side effects for a wide varietyof diseases.

SUMMARY OF THE INVENTION

This invention provides an isolated nucleic acid encoding a mammalianSNORF25 receptor.

This invention further provides a purified mammalian SNORF25 receptorprotein.

This invention also provides a vector comprising a nucleic acid inaccordance with this invention.

This invention still further provides a cell comprising a vector inaccordance with this invention.

This invention additionally provides a membrane preparation isolatedfrom a cell in accordance with this invention.

Furthermore, this invention provides a nucleic acid probe comprising atleast 15 nucleotides, which probe specifically hybridizes with a nucleicacid encoding a mammalian SNORF25 receptor, wherein the probe has asequence complementary to a unique sequence present within one of thetwo strands of the nucleic acid encoding the mammalian SNORF25 receptorcontained in plasmid pEXJT3T7-hSNORF25 (ATCC Accession No. 203495).

This invention further provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a mammalian SNORF25 receptor, wherein the probe has a sequencecomplementary to a unique sequence present within (a) the nucleic acidsequence shown in FIGS. 1A-1B (SEQ ID NO: 1) or (b) the reversecomplement thereof.

This invention provides an antisense oligonucleotide having a sequencecapable of specifically hybridizing to RNA encoding a mammalian SNORF25receptor, so as to prevent translation of such RNA.

This invention further provides an antisense oligonucleotide having asequence capable of specifically hybridizing to genomic DNA encoding amammalian SNORF25 receptor, so as to prevent transcription of suchgenomic DNA.

This invention also provides an antibody capable of binding to amammalian SNORF25 receptor encoded by a nucleic acid in accordance withthis invention.

Moreover, this invention provides an agent capable of competitivelyinhibiting the binding of an antibody in accordance with this inventionto a mammalian SNORF25 receptor.

This invention still further provides a pharmaceutical compositioncomprising (a) an amount of an oligonucleotide in accordance with thisinvention capable of passing through a cell membrane and effective toreduce expression of a mammalian SNORF25 receptor and (b) apharmaceutically acceptable carrier capable of passing through the cellmembrane.

This invention also provides a pharmaceutical composition whichcomprises an amount of an antibody in accordance with this inventioneffective to block binding of a ligand to a human SNORF25 receptor and apharmaceutically acceptable carrier.

This invention further provides a transgenic, nonhuman mammal expressingDNA encoding a mammalian SNORF25 receptor in accordance with thisinvention.

This invention still further provides a transgenic, nonhuman mammalcomprising a homologous recombination knockout of a native mammalianSNORF25 receptor.

This invention further provides a transgenic, nonhuman mammal whosegenome comprises antisense DNA complementary to DNA encoding a mammalianSNORF25 receptor in accordance with this invention so placed within suchgenome as to be transcribed into antisense mRNA which is complementaryto and hybridizes with mRNA encoding the mammalian SNORF25 receptor soas to reduce translation of of such mRNA and expression of suchreceptor.

This invention provides a process for identifying a chemical compoundwhich specifically binds to a mammalian SNORF25 receptor which comprisescontacting cells containing DNA encoding, and expressing on their cellsurface, the mammalian SNORF25 receptor, wherein such cells do notnormally express the mammalian SNORF25 receptor, with the compound underconditions suitable for binding, and detecting specific binding of thechemical compound to the mammalian SNORF25 receptor.

This invention further provides a process for identifying a chemicalcompound which specifically binds to a mammalian SNORF25 receptor whichcomprises contacting a membrane preparation from cells containing DNAencoding, and expressing on their cell surface, the mammalian SNORF25receptor, wherein such cells do not normally express the mammalianSNORF25 receptor, with the compound under conditions suitable forbinding, and detecting specific binding of the chemical compound to themammalian SNORF25 receptor.

This invention still further provides a process involving competitivebinding for identifying a chemical compound which specifically binds toa mammalian SNORF25 receptor which comprises separately contacting cellsexpressing on their cell surface the mammalian SNORF25 receptor, whereinsuch cells do not normally express the mammalian SNORF25 receptor, withboth the chemical compound and a second chemical compound known to bindto the receptor, and with only the second chemical compound, underconditions suitable for binding of such compounds to the receptor, anddetecting specific binding of the chemical compound to the mammalianSNORF25 receptor, a decrease in the binding of the second chemicalcompound to the mammalian SNORF25 receptor in the presence of thechemical compound being tested indicating that such chemical compoundbinds to the mammalian SNORF25 receptor.

This invention further provides a process involving competitive bindingfor identifying a chemical compound which specifically binds to amammalian SNORF25 receptor which comprises separately contacting amembrane preparation from cells expressing on their cell surface themammalian SNORF25 receptor, wherein such cells do not normally expressthe mammalian SNORF25 receptor, with both the chemical compound and asecond chemical compound known to bind to the receptor, and with onlythe second chemical compound, under conditions suitable for binding ofsuch compounds to the receptor, and detecting specific binding of thechemical compound to the mammalian SNORF25 receptor, a decrease in thebinding of the second chemical compound to the mammalian SNORF25receptor in the presence of the chemical compound indicating that thechemical compound binds to the mammalian SNORF25 receptor.

This invention further provides a compound identified by one of theprocesses of this invention.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a mammalian SNORF25 receptor to identifya compound which specifically binds to the mammalian SNORF25 receptor,which comprises (a)contacting cells transfected with, and expressing,DNA encoding the mammalian SNORF25 receptor with a compound known tobind specifically to the mammalian SNORF25 receptor; (b) contacting thecells of step (a) with the plurality of compounds not known to bindspecifically to the mammalian SNORF25 receptor, under conditionspermitting binding of compounds known to bind to the mammalian SNORF25receptor; (c) determining whether the binding of the compound known tobind to the mammalian SNORF25 receptor is reduced in the presence of theplurality of compounds, relative to the binding of the compound in theabsence of the plurality of compounds; and if so (d) separatelydetermining the binding to the mammalian SNORF25 receptor of eachcompound included in the plurality of compounds, so as to therebyidentify any compound included therein which specifically binds to themammalian SNORF25 receptor.

This invention further provides a method of screening a plurality ofchemical compounds not known to bind to a mammalian SNORF25 receptor toidentify a compound which specifically binds to the mammalian SNORF25receptor, which comprises (a) contacting a membrane preparation fromcells transfected with, and expressing, DNA encoding the mammalianSNORF25 receptor with the plurality of compounds not known to bindspecifically to the mammalian SNORF25 receptor under conditionspermitting binding of compounds known to bind to the mammalian SNORF25receptor; (b) determining whether the binding of a compound known tobind to the mammalian SNORF25 receptor is reduced in the presence of anycompound within the plurality of compounds, relative to the binding ofthe compound in the absence of the plurality of compounds; and if so (c)separately determining the binding to the mammalian SNORF25 receptor ofeach compound included in the plurality of compounds, so as to therebyidentify any compound included therein which specifically binds to themammalian SNORF25 receptor.

This invention also provides a method of detecting expression of amammalian SNORF25 receptor by detecting the presence of mRNA coding forthe mammalian SNORF25 receptor which comprises obtaining total mRNA fromthe cell and contacting the mRNA so obtained with a nucleic acid probeaccording to this invention under hybridizing conditions, detecting thepresence of mRNA hybridized to the probe, and thereby detecting theexpression of the mammalian SNORF25 receptor by the cell.

This invention further provides a method of detecting the presence of amammalian SNORF25 receptor on the surface of a cell which comprisescontacting the cell with an antibody according to this invention underconditions permitting binding of the antibody to the receptor, detectingthe presence of the antibody bound to the cell, and thereby detectingthe presence of the mammalian SNORF25 receptor on the surface of thecell.

This invention still further provides a method of determining thephysiological effects of varying levels of activity of mammalian SNORF25receptors which comprises producing a transgenic, nonhuman mammal inaccordance with this invention whose levels of mammalian SNORF25receptor activity are varied by use of an inducible promoter whichregulates mammalian SNORF25 receptor expression.

This invention additionally provides a method of determining thephysiological effects of varying levels of activity of mammalian SNORF25receptors which comprises producing a panel of transgenic, nonhumanmammals in accordance with this invention each expressing a differentamount of mammalian SNORF25 receptor.

Moreover, this invention provides a method for identifying an antagonistcapable of alleviating an abnormality wherein the abnormality isalleviated by decreasing the activity of a mammalian SNORF25 receptorcomprising administering a compound to a transgenic, nonhuman mammalaccording to this invention, and determining whether the compoundalleviates any physiological and/or behavioral abnormality displayed bythe transgenic, nonhuman mammal as a result of overactivity of amammalian SNORF25 receptor, the alleviation of such an abnormalityidentifying the compound as an antagonist.

This invention also provides an antagonist identified by the precedingmethod.

This invention further provides a composition, e.g. a pharmaceuticalcomposition, comprising an antagonist according to this invention and acarrier, e.g. a pharmaceutically acceptable carrier.

This invention additionally provides a method of treating an abnormalityin a subject wherein the abnormality is alleviated by decreasing theactivity of a mammalian SNORF25 receptor which comprises administeringto the subject an effective amount of the pharmaceutical compositionaccording to this invention so as to thereby treat the abnormality.

In addition, this invention provides a method for identifying an agonistcapable of alleviating an abnormality in a subject wherein theabnormality is alleviated by increasing the activity of a mammalianSNORF25 receptor comprising administering a compound to a transgenic,nonhuman mammal according to this invention, and determining whether thecompound alleviates any physiological and/or behavioral abnormalitydisplayed by the transgenic, nonhuman mammal, the alleviation of such anabnormality identifying the compound as an agonist.

This invention further provides an agonist identified by the precedingmethod.

This invention still further provides a composition, e.g. apharmaceutical composition, comprising an agonist according to thisinvention and a carrier, e.g. pharmaceutically acceptable carrier.

Moreover, this invention provides a method of treating an abnormality ina subject wherein the abnormality is alleviated by increasing theactivity of a mammalian SNORF25 receptor which comprises administeringto the subject an effective amount of the pharmaceutical compositionaccording to this invention so as to thereby treat the abnormality.

Yet further, this invention provides a method for diagnosing apredisposition to a disorder associated with the activity of a specificmammalian allele which comprises: (a) obtaining DNA of subjectssuffering from the disorder; (b) performing a restriction digest of theDNA with a panel of restriction enzymes; (c) electrophoreticallyseparating the resulting DNA fragments on a sizing gel; (d) contactingthe resulting gel with a nucleic acid probe capable of specificallyhybridizing with a unique sequence included within the sequence of anucleic acid molecule encoding a mammalian SNORF25 receptor and labeledwith a detectable marker; (e) detecting labeled bands which havehybridized to the DNA encoding a mammalian SNORF25 receptor to create aunique band pattern specific to the DNA of subjects suffering from thedisorder; (f) repeating steps (a)-(e) with DNA obtained for diagnosisfrom subjects not yet suffering from the disorder; and (g) comparing theunique band pattern specific to the DNA of subjects suffering from thedisorder from step (e) with the band pattern from step (f) for subjectsnot yet suffering from the disorder so as to determine whether thepatterns are the same or different and thereby diagnose predispositionto the disorder if the patterns are the same.

This invention also provides a method of preparing a purified mammalianSNORF25 receptor according to the invention which comprises: (a)culturing cells which express the mammalian SNORF25 receptor; (b)recovering the mammalian SNORF25 receptor from the cells; and (c)purifying the mammalian SNORF25 receptor so recovered.

This invention further provides a method of preparing the purifiedmammalian SNORF25 receptor according to the invention which comprises:(a) inserting a nucleic acid encoding the mammalian SNORF25 receptorinto a suitable expression vector; (b) introducing the resulting vectorinto a suitable host cell; (c) placing the resulting host cell insuitable conditions permitting the production of the mammalian SNORF25receptor; (d) recovering the mammalian SNORF25 receptor so produced; andoptionally (e) isolating and/or purifying the mammalian SNORF25 receptorso recovered.

Furthermore, this invention provides a process for determining whether achemical compound is a mammalian SNORF25 receptor agonist whichcomprises contacting cells transfected with and expressing DNA encodingthe mammalian SNORF25 receptor with the compound under conditionspermitting the activation of the mammalian SNORF25 receptor, anddetecting any increase in mammalian SNORF25 receptor activity, so as tothereby determine whether the compound is a mammalian SNORF25 receptoragonist.

This invention also provides a process for determining whether achemical compound is a mammalian SNORF25 receptor antagonist whichcomprises contacting cells transfected with and expressing DNA encodingthe mammalian SNORF25 receptor with the compound in the presence of aknown mammalian SNORF2S receptor agonist, under conditions permittingthe activation of the mammalian SNORF25 receptor, and detecting anydecrease in mammalian SNORF25 receptor activity, so as to therebydetermine whether the compound is a mammalian SNORF25 receptorantagonist.

This invention still further provides a composition, for example apharmaceutical composition, which comprises an amount of a mammalianSNORF25 receptor agonist determined by a process according to thisinvention effective to increase activity of a mammalian SNORF25 receptorand a carrier, for example, a pharmaceutically acceptable carrier.

Also, this invention provides a composition, for example apharmaceutical composition, which comprises an amount of a mammalianSNORF25 receptor antagonist determined by a process according to thisinvention effective to reduce activity of a mammalian SNORF25 receptorand a carrier, for example, a pharmaceutically acceptable carrier.

This invention moreover provides a process for determining whether achemical compound specifically binds to and activates a mammalianSNORF25 receptor, which comprises contacting cells producing a secondmessenger response and expressing on their cell surface the mammalianSNORF25 receptor, wherein such cells do not normally express themammalian SNORF25 receptor, with the chemical compound under conditionssuitable for activation of the mammalian SNORF25 receptor, and measuringthe second messenger response in the presence and in the absence of thechemical compound, a change, e.g. an increase, in the second messengerresponse in the presence of the chemical compound indicating that thecompound activates the mammalian SNORF25 receptor.

This invention still further provides a process for determining whethera chemical compound specifically binds to and inhibits activation of amammalian SNORF25 receptor, which comprises separately contacting cellsproducing a second messenger response and expressing on their cellsurface the mammalian SNORF25 receptor, wherein such cells do notnormally express the mammalian SNORF25 receptor, with both the chemicalcompound and a second chemical compound known to activate the mammalianSNORF25 receptor, and with only the second chemical compound, underconditions suitable for activation of the mammalian SNORF25 receptor,and measuring the second messenger response in the presence of only thesecond chemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change, e.g. increase, inthe second messenger response in the presence of both the chemicalcompound and the second chemical compound than in the presence of onlythe second chemical compound indicating that the chemical compoundinhibits activation of the mammalian SNORF25 receptor.

Further, this invention provides a compound determined by a processaccording to the invention and a composition, for example, apharmaceutical composition, which comprises an amount of a mammalianSNORF25 receptor agonist determined to be such by a process according tothe invention, effective to increase activity of the mammalian SNORF25receptor and a carrier, for example, a pharmaceutically acceptablecarrier.

This invention also provides a composition, for example, apharmaceutical composition, which comprises an amount of a mammalianSNORF25 receptor antagonist determined to be such by a process accordingto the invention, effective to reduce activity of the mammalian SNORF25receptor and a carrier, for example, a pharmaceutically acceptablecarrier.

This invention yet further provides a method of screening a plurality ofchemical compounds not known to activate a mammalian SNORF25 receptor toidentify a compound which activates the mammalian SNORF25 receptor whichcomprises: (a) contacting cells transfected with and expressing themammalian SNORF25 receptor with the plurality of compounds not known toactivate the mammalian SNORF25 receptor, under conditions permittingactivation of the mammalian SNORF25 receptor; (b) determining whetherthe activity of the mammalian SNORF25 receptor is increased in thepresence of one or more the compounds; and if so (c) separatelydetermining whether the activation of the mammalian SNORF25 receptor isincreased by any compound included in the plurality of compounds, so asto thereby identify each compound which activates the mammalian SNORF25receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to inhibit the activation of a mammalian SNORF25receptor to identify a compound which inhibits the activation of themammalian SNORF25 receptor, which comprises: (a) contacting cellstransfected with and expressing the mammalian SNORF25 receptor with theplurality of compounds in the presence of a known mammalian SNORF25receptor agonist, under conditions permitting activation of themammalian SNORF25 receptor; (b) determining whether the extent or amountof activation of the mammalian SNORF25 receptor is reduced in thepresence of one or more of the compounds, relative to the extent oramount of activation of the mammalian SNORF25 receptor in the absence ofsuch one or more compounds; and if so (c) separately determining whethereach such compound inhibits activation of the mammalian SNORF25 receptorfor each compound included in the plurality of compounds, so as tothereby identify any compound included in such plurality of compoundswhich inhibits the activation of the mammalian SNORF25 receptor.

This invention also provides a composition, for example a pharmaceuticalcomposition, comprising a compound identified by a method according tothis invention in an amount effective to increase mammalian SNORF25receptor activity and a carrier, for example, a pharmaceuticallyacceptable carrier.

This invention still further provides a composition, for example, apharmaceutical composition, comprising a compound identified by a methodaccording to this invention in an amount effective to decrease mammalianSNORF25 receptor activity and a carrier, for example, a pharmaceuticallyacceptable carrier.

Furthermore, this invention provides a method of treating an abnormalityin a subject wherein the abnormality is alleviated by increasing theactivity of a mammalian SNORF25 receptor which comprises administeringto the subject a compound which is a mammalian SNORF25 receptor agonistin an amount effective to treat the abnormality.

This invention additionally provides a method of treating an abnormalityin a subject wherein the abnormality is alleviated by decreasing theactivity of a mammalian SNORF25 receptor which comprises administeringto the subject a compound which is a mammalian SNORF25 receptorantagonist in an amount effective to treat the abnormality.

This invention also provides a process for making a composition ofmatter which specifically binds to a mammalian SNORF25 receptor whichcomprises identifying a chemical compound using a process in accordancewith this invention and then synthesizing the chemical compound or anovel structural and functional analog or homolog thereof.

This invention further provides a process for preparing a composition,for example, a pharmaceutical composition which comprises admixing acarrier, for example, a pharmaceutically acceptable carrier, and apharmaceutically effective amount of a chemical compound identified by aprocess in accordance with this invention or a novel structural andfunctional analog or homolog thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B

Nucleotide sequence including sequence encoding a human SNORF25 receptor(SEQ ID NO: 1). Putative open reading frames including the shortest openreading frame are indicated by underlining one start (ATG) codon (atpositions 61-63) and the stop codon (at positions 1066-1068). Inaddition, partial 5′ and 3′ untranslated sequences are shown.

FIGS. 2A-2B

Deduced amino acid sequence (SEQ ID NO: 2) of the human SNORF25 receptorencoded by the longest open reading frame indicated in the nucleotidesequence shown in FIGS. 1A-1B (SEQ ID NO: 1). The seven putativetransmembrane (TM) regions are underlined.

FIGS. 3A-3B

Nucleotide sequence including sequence encoding a rat SNORF25 receptor(SEQ ID NO: 3). Putative open reading frames including the shortest openreading frame are indicated by underlining one start (ATG) codon (atpositions 49-51) and the stop codon (at positions 1054-1056). Inaddition, partial 5′ and 3′ untranslated sequences are shown.

FIGS. 4A-4B

Deduced amino acid sequence (SEQ ID NO: 4) of the rat SNORF25 receptorencoded by the longest open reading frame indicated in the nucleotidesequence shown in FIGS. 3A-3B (SEQ. ID NO: 3). The seven putativetransmembrane (TM) regions are underlined.

FIG. 5

Comparison of basal cAMP levels of SNORF25-and mock-transfected CHOcells. SNORF25 or empty vector (mock) DNA was transfected into CHO cellsas described in Materials and Methods. The transfectants were platedinto 96-well plates, and assayed for cAMP release as described. Theresults of a representative experiment are shown.

FIG. 6

Modulation of cAMP release by ATRA, vitamin A1 and forskolin inSNORF25-expressing and mock-transfected CHO cells. The transfectantswere plated into 96-well plates, challenged with 10 μM concentrations ofdrugs and assayed for cAMP release as described. The results of arepresentative experiment involving known cyclase stimulatory receptorsare shown. Results are means ±S.E.M of triplicate determinations withthe exception of vitamin A₁ which is a single point. Results arenormalized to % basal cAMP release.

FIG. 7

Specificity of ATRA cAMP response in Cos-7 cells. The transfectants wereplated into 96-well plates, challenged with 10 μM concentrations of ATRAand assayed for cAMP release as described. The results of arepresentative experiment are shown. Results are means ±S.E.M oftriplicate determinations.

FIG. 8

ATRA Dose-response curve in transiently-transfected Cos-7 cells. Arepresentative example of dose-response effect of ATRA to increase cAMPrelease in SNORF25- (▪) and mock- (□) transfected cells.

FIGS. 9A-9C

Stimulation of CFTR by ATRA in ooctyes expressing SNORF25. Voltage clamprecording from oocyte previously injected with SNORF25 receptor mRNA andCFTR (FIG. 9A), and from control (CFTR alone) oocyte (FIG. 9B).Application of epinephrine (1 μM) evokes a similar current in otheroocytes expressing the B2 adrenergic receptor (B2AR) and CFTR (FIG. 9C).Holding potential was −70 mV for all recordings.

FIG. 10

Mean current amplitudes stimulated by ATRA (10 μM) in control (CFTRalone) ooctyes (n=16) and oocytes injected with mRNA encoding SNORF25and CFTR (n=17).

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a recombinant nucleic acid comprising a nucleicacid encoding a mammalian SNORF25 receptor, wherein the mammalianreceptor-encoding nucleic acid hybridizes under high stringencyconditions to (a) a nucleic acid encoding a human SNORF25 receptor andhaving a sequence identical to the sequence of the human SNORF25receptor-encoding nucleic acid contained in plasmid pEXJT3T7-hSNORF25(ATCC Accession No. 203495) or (b) a nucleic acid encoding a rat SNORF25receptor and having a sequence identical to the sequence of the ratSNORF25 receptor-encoding nucleic acid contained in plasmidpcDNA3.1-rSNORF25 (ATCC Accession No. 203494).

This invention further provides a recombinant nucleic acid comprising anucleic acid encoding a human SNORF25 receptor, wherein the humanSNORF25 receptor comprises an amino acid sequence identical to thesequence of the human SNORF25 receptor encoded by the shortest openreading frame indicated in FIGS. 1A-1B (SEQ ID NO: 1).

This invention also provides a recombinant nucleic acid comprising anucleic acid encoding a rat SNORF25 receptor, wherein the rat SNORF25receptor comprises an amino acid sequence identical to the sequence ofthe rat SNORF25 receptor encoded by the shortest open reading frameindicated in FIGS. 3A-3B (SEQ ID NO: 3).

Plasmid pEXJT3T7-hSNORF25 and plasmid pcDNA3.1-rSNORF25 were bothdeposited on Nov. 24, 1998, with the American Type Culture Collection(ATCC), 10801 University Blvd., Manassas, Va. 20110-2209, U.S.A. underthe provisions of the Budapest Treaty for the International Recognitionof the Deposit of Microorganisms for the Purposes of Patent Procedureand were accorded ATCC Accession Nos. 203495 and 203494, respectively.

Hybridization methods are well known to those of skill in the art. Forpurposes of this invention, hybridization under high stringencyconditions means hybridization performed at 40° C. in a hybridizationbuffer containing 50% formamide, 5× SSC, 7 mM Tris, 1× Denhardt's, 25μg/ml salmon sperm DNA; wash at 50° C. in 0.1× SSC, 0.1% SDS.

Throughout this application, the following standard abbreviations areused to indicate specific nucleotide bases:

A=adenine

G=guanine

C=cytosine

T=thymine

M=adenine or cytosine

R=adenine or guanine

W=adenine or thymine

S=cytosine or guanine

Y=cytosine or thymine

K=guanine or thymine

V=adenine, cytosine, or guanine (not thymine)

H=adenine, cytosine, or thymine (not cytosine)

B=cytosine, guanine, or thymine (not adenine)

N=adenine, cytosine, guanine, or thymine (or other modified base such asinosine)

I=inosine

Furthermore, the term “agonist” is used throughout this application toindicate any peptide or non-peptidyl compound which increases theactivity of any of the polypeptides of the subject invention. The term“antagonist” is used throughout this application to indicate any peptideor non-peptidyl compound which decreases the activity of any of thepolypeptides of the subject invention.

Furthermore, as used herein, the phrase “pharmaceutically acceptablecarrier” means any of the standard pharmaceutically acceptable carriers.Examples include, but are not limited to, phosphate buffered saline,physiological saline, water, and emulsions, such as oil/water emulsions.

It is possible that the mammalian SNORF25 receptor gene contains intronsand furthermore, the possibility exists that additional introns couldexist in coding or non-coding regions. In addition, spliced form(s) ofmRNA may encode additional amino acids either upstream of the currentlydefined starting methionine or within the coding region. Further, theexistence and use of alternative exons is possible, whereby the mRNA mayencode different amino acids within the region comprising the exon. Inaddition, single amino acid substitutions may arise via the mechanism ofRNA editing such that the amino acid sequence of the expressed proteinis different than that encoded by the original gene. (Burns, et al.,1996; Chu, et al., 1996). Such variants may exhibit pharmacologicproperties differing from the polypeptide encoded by the original gene.

This invention provides splice variants of the mammalian SNORF25receptors disclosed herein. This invention further provides alternatetranslation initiation sites and alternately spliced or edited variantsof nucleic acids encoding the SNORF25 receptors in accordance with thisinvention.

This invention also contemplates recombinant nucleic acids whichcomprise nucleic acids encoding naturally occurring allelic variants ofthe SNORF25 receptors disclosed herein.

The nucleic acids of the subject invention also include nucleic acidanalogs of the human SNORF25 receptor genes, wherein the human SNORF25receptor gene comprises the nucleic acid sequence shown in FIGS. 1A-1Bor contained in plasmid pEXJT3T7-hSNORF25 (ATCC Accession No. 203495).Nucleic acid analogs of the human SNORF25 receptor genes differ from thehuman SNORF25 receptor genes described herein in terms of the identityor location of one or more nucleic acid bases (deletion analogscontaining less than all of the nucleic acid bases shown in FIGS. 1A-1Bor contained in plasmid pEXJT3T7-hSNORF25 (ATCC Accession No. 203495),substitution analogs wherein one or more nucleic acid bases shown inFIGS. 1A-1B or contained in plasmid pEXJT3T7-hSNORF25 (ATCC AccessionNo. 203495), are replaced by other nucleic acid bases, and additionanalogs, wherein one or more nucleic acid bases are added to a terminalor medial portion of the nucleic acid sequence) and which encodeproteins which share some or all of the properties of the proteinsencoded by the nucleic acid sequences shown in FIGS. 1A-1B or containedin plasmid pEXJT3T7-hSNORF25 (ATCC Accession No. 203495). In oneembodiment of the present invention, the nucleic acid analog encodes aprotein which has an amino acid sequence identical to that shown inFIGS. 2A-2B or encoded by the nucleic acid sequence contained in plasmidpEXJT3T7-hSNORF25 (ATCC Accession No. 203495). In another embodiment,the nucleic acid analog encodes a protein having an amino acid sequencewhich differs from the amino acid sequences shown in FIGS. 2A-2B orencoded by the nucleic acid contained in plasmid pEXJT3T7-hSNORF25 (ATCCAccession No. 203495). In a further embodiment, the protein encoded bythe nucleic acid analog has a function which is the same as the functionof the receptor proteins having the amino acid sequence shown in FIGS.2A-2B. In another embodiment, the function of the protein encoded by thenucleic acid analog differs from the function of the receptor proteinhaving the amino acid sequence shown in FIGS. 2A-2B. In anotherembodiment, the variation in the nucleic acid sequence occurs within thetransmembrane (TM) region of the protein. In a further embodiment, thevariation in the nucleic acid sequence occurs outside of the TM region.

The nucleic acids of the subject invention also include nucleic acidanalogs of the rat SNORF25 receptor genes, wherein the rat SNORF25receptor gene comprises the nucleic acid sequence shown in FIGS. 3A-3Bor contained in plasmid pcDNA3.1-rSNORF25 (ATCC Accession No. 203494).Nucleic acid analogs of the rat SNORF25 receptor genes differ from therat SNORF25 receptor genes described herein in terms of the identity orlocation of one or more nucleic acid bases (deletion analogs containingless than all of the nucleic acid bases shown in FIGS. 3A-3B orcontained in plasmid pcDNA3.1-rSNORF25 (ATCC Accession No. 203494)substitution analogs wherein one or more nucleic acid bases shown inFIGS. 3A-3B or contained in plasmid pcDNA3.1-rSNORF25 (ATCC AccessionNo. 203494), are replaced by other nucleic acid bases, and additionanalogs, wherein one or more nucleic acid bases are added to a terminalor medial portion of the nucleic acid sequence) and which encodeproteins which share some or all of the properties of the proteinsencoded by the nucleic acid sequences shown in FIG. 3A-3B or containedin plasmid pcDNA3.1-rSNORF25 (ATCC Accession No. 203494). In oneembodiment of the present invention, the nucleic acid analog encodes aprotein which has an amino acid sequence identical to that shown inFIGS. 4A-4B or encoded by the nucleic acid sequence contained in plasmidpcDNA3.1-rSNORF25 (ATCC Accession No. 203494). In another embodiment,the nucleic acid analog encodes a protein having an amino acid sequencewhich differs from the amino acid sequences shown in FIGS. 4A-4B orencoded by the nucleic acid contained in plasmid pcDNA3.1-rSNORF25 (ATCCAccession No. 203494). In a further embodiment, the protein encoded bythe nucleic acid analog has a function which is the same as the functionof the receptor proteins having the amino acid sequence shown in FIGS.4A-4B. In another embodiment, the function of the protein encoded by thenucleic acid analog differs from the function of the receptor proteinhaving the amino acid sequence shown in FIGS. 4A-4B. In anotherembodiment, the variation in the nucleic acid sequence occurs within thetransmembrane (TM) region of the protein. In a further embodiment, thevariation in the nucleic acid sequence occurs outside of the TM region.

This invention provides the above-described isolated nucleic acid,wherein the nucleic acid is DNA. In an embodiment, the DNA is cDNA. Inanother embodiment, the DNA is genomic DNA. In still another embodiment,the nucleic acid is RNA. Methods for production and manipulation ofnucleic acid molecules are well known in the art.

This invention further provides nucleic acid which is degenerate withrespect to the DNA encoding any of the polypeptides described herein. Inan embodiment, the nucleic acid comprises a nucleotide sequence which isdegenerate with respect to the nucleotide sequence shown in FIGS. 1A-1B(SEQ ID NO: 1) or the nucleotide sequence contained in the plasmidpEXJT3T7-hSNORF25 (ATCC Accession No. 203495), that is, a nucleotidesequence which is translated into the same amino acid sequence.

This invention further provides nucleic acid which is degenerate withrespect to the DNA encoding any of the polypeptides described herein. Inan embodiment, the nucleic acid comprises a nucleotide sequence which isdegenerate with respect to the nucleotide sequence shown in FIGS. 3A-3B(SEQ ID NO: 3) or the nucleotide sequence contained in the plasmidpcDNA3.1-rSNORF25 (ATCC Accession No. 203494), that is, a nucleotidesequence which is translated into the same amino acid sequence.

This invention also encompasses DNAs and cDNAs which encode amino acidsequences which differ from those of the polypeptides according to thisinvention, but which should not produce phenotypic changes. Alternately,this invention also encompasses DNAs, cDNAs, and RNAs which hybridizewith the DNA, cDNA, and RNA according to the subject invention.Hybridization methods are well known to those of skill in the art.

The nucleic acids according to the subject invention also includenucleic acid molecules coding for polypeptide analogs, fragments orderivatives of antigenic polypeptides which differ fromnaturally-occurring forms in terms of the identity or location of one ormore amino acid residues (deletion analogs containing less than all ofthe residues specified for the protein, substitution analogs wherein oneor more residues specified are replaced by other residues and additionanalogs wherein one or more amino acid residues is added to a terminalor medial portion of the polypeptides) and which share some or allproperties of naturally-occurring forms. These molecules include: theincorporation of codons “preferred” for expression by selectednon-mammalian hosts; the provision of sites for cleavage by restrictionendonuclease enzymes; and the provision of additional initial, terminalor intermediate DNA sequences that facilitate construction of readilyexpressed vectors. The creation of polypeptide analogs is well known tothose of skill in the art (Spurney, R. F. et al. (1997); Fong, T. M. etal. (1995); Underwood, D. J. et al. (1994); Graziano, M. P. et al.(1996); Guan X. M. et al. (1995)).

The modified polypeptides according to this invention may be transfectedinto cells either transiently or stably using methods well-known in theart, examples of which are disclosed herein. This invention alsoprovides binding assays using the modified polypeptides, in which thepolypeptide is expressed either transiently or in stable cell lines.This invention further provides a compound identified using a modifiedpolypeptide in a binding assay such as the binding assays describedherein.

The nucleic acids described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide and as products for the large scale synthesis of thepolypeptides by a variety of recombinant techniques. The nucleic acidmolecule is useful for generating new cloning and expression vectors,transformed and transfected prokaryotic and eukaryotic host cells, andnew and useful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

This invention also provides an isolated nucleic acid encoding specieshomologs of the SNORF25 receptors encoded by the nucleic acid sequenceshown in FIGS. 1A-1B (SEQ ID NO: 1) or encoded by the plasmidpEXJT3T7-hSNORF2S (ATCC Accession No. 203495). In one embodiment, thenucleic acid encodes a mammalian SNORF25 receptor homolog which hassubstantially the same amino acid sequence as does the SNORF25 receptorencoded by the plasmid pEXJT3T7-hSNORF25 (ATCC Accession No. 203495). Inanother embodiment, the nucleic acid encodes a mammalian SNORF25receptor homolog which has above 75% amino acid identity to the SNORF25receptor encoded by the plasmid pEXJT3T7-hSNORF25 (ATCC Accession No.203495); preferably above 85% amino acid identity to the SNORF25receptor encoded by the plasmid pEXJT3T7-hSNORF25 (ATCC Accession No.203495); most preferably above 95% amino acid identity to the SNORF25receptor encoded by the plasmid pEXJT3T7-hSNORF25 (ATCC Accession No.203495). In another embodiment, the mammalian SNORF25 receptor homologhas above 70% nucleic acid identity to the SNORF25 receptor genecontained in plasmid pEXJT3T7-hSNORF25 (ATCC Accession No. 203495);preferably above 80% nucleic acid identity to the SNORF25 receptor genecontained in the plasmid pEXJT3T7-hSNORF25 (ATCC Accession No. 203495);more preferably above 90% nucleic acid identity to the SNORF25 receptorgene contained in the plasmid pEXJT3T7-hSNORF25 (ATCC Accession No.203495). Examples of methods for isolating and purifying specieshomologs are described elsewhere (e.g., U.S. Pat. No. 5,602,024,WO94/14957, WO97/26853, WO98/15570).

This invention also provides an isolated nucleic acid encoding specieshomologs of the SNORF25 receptors encoded by the nucleic acid sequenceshown in FIGS. 3A-3B (SEQ ID NO: 3) or encoded by the plasmidpcDNA3.1-rSNORF25 (ATCC Accession No. 203494). In one embodiment, thenucleic acid encodes a mammalian SNORF25 receptor homolog which hassubstantially the same amino acid sequence as does the SNORF25 receptorencoded by the plasmid pcDNA3.1-rSNORF25 (ATCC Accession No. 203494). Inanother embodiment, the nucleic acid encodes a mammalian SNORF25receptor homolog which has above 75% amino acid identity to the SNORF25receptor encoded by the plasmid pcDNA3.1-rSNORF25 (ATCC Accession No.203494); preferably above 85% amino acid identity to the SNORF25receptor encoded by the plasmid pcDNA3.1-rSNORF25 (ATCC Accession No.203494); most preferably above 95% amino acid identity to the SNORF25receptor encoded by the plasmid pcDNA3.1-rSNORF25 (ATCC Accession No.203494). In another embodiment, the mammalian SNORF25 receptor homologhas above 70% nucleic acid identity to the SNORF25 receptor genecontained in plasmid pcDNA3.1-rSNORF25 (ATCC Accession No. 203494);preferably above 80% nucleic acid identity to the SNORF25 receptor genecontained in the plasmid pcDNA3.1-rSNORF25 (ATCC Accession No. 203494);more preferably above 90% nucleic acid identity to the SNORF25 receptorgene contained in the plasmid pcDNA3.1-rSNORF25 (ATCC Accession No.203494).

This invention provides an isolated nucleic acid encoding a modifiedmammalian SNORF25 receptor, which differs from a mammalian SNORF25receptor by having an amino acid(s) deletion, replacement, or additionin the third intracellular domain.

This invention provides an isolated nucleic acid encoding a mammalianSNORF25 receptor. In one embodiment, the nucleic acid is DNA. In anotherembodiment, the DNA is cDNA. In another embodiment, the DNA is genomicDNA. In another embodiment, the nucleic acid is RNA. In anotherembodiment, the mammalian SNORF25 receptor is a human SNORF25 receptor.In another embodiment, the human SNORF25 receptor has an amino acidsequence identical to that encoded by the plasmid pEXJT3 T7-hSNORF25(ATCC Accession No. 203495). In another embodiment, the human SNORF25receptor has an amino acid sequence identical to the amino acid sequenceshown in FIGS. 2A-2B (SEQ ID NO: 2).

In an embodiment, the mammalian SNORF25 receptor is a rat SNORF25receptor. In another embodiment, the rat SNORF25 receptor has an aminoacid sequence identical to that encoded by the plasmid pcDNA3.1-rSNORF25(ATCC Accession No. 203494) . In another embodiment, the rat SNORF25receptor has an amino acid sequence identical to the amino acid sequenceshown in FIGS. 4A-4B (SEQ ID NO: 4).

This invention provides a purified mammalian SNORF25 receptor protein.In one embodiment, the SNORF25 receptor protein is a human SNORF25receptor protein. In a further embodiment, the SNORF25 receptor proteinis a rat SNORF25 receptor protein.

This invention provides a vector comprising a nucleic acid in accordancewith this invention. This invention further provides a vector adaptedfor expression in a cell which comprises the regulatory elementsnecessary for expression of the nucleic acid in the cell operativelylinked to the nucleic acid encoding the receptor so as to permitexpression thereof, wherein the cell is a bacterial, amphibian, yeast,insect or mammalian cell. In one embodiment, the vector is abaculovirus. In another embodiment, the vector is a plasmid.

This invention provides a plasmid designated pEXJT3T7-hSNORF25 (ATCCAccession No. 203495). This invention also provides a plasmid designatedpcDNA3.1-rSNORF25 (ATCC Accession No. 203494).

This invention further provides any vector or plasmid which comprisesmodified untranslated sequences, which are beneficial for expression indesired host cells or for use in binding or functional assays. Forexample, a vector or plasmid with untranslated sequences of varyinglengths may express differing amounts of the polypeptide depending uponthe host cell used. In an embodiment, the vector or plasmid comprisesthe coding sequence of the polypeptide and the regulatory elementsnecessary for expression in the host cell.

This invention provides a cell comprising a vector in accordance withthis invention. In one embodiment, the cell is a non-mammalian cell. Inone embodiment, the non-mammalian cell is a Xenopus oocyte cell or aXenopus melanophore cell. In another embodiment, the cell is a mammaliancell. In another embodiment, the cell is a COS-7 cell, a 293 humanembryonic kidney cell, a NIH-3T3 cell, a LM(tk−) cell, a mouse Y1 cell,or a CHO cell. In another embodiment, the cell is an insect cell. Inanother embodiment, the insect cell is an Sf9 cell, an Sf21 cell or aTrichoplusia ni 5B-4 cell.

This invention provides a membrane preparation isolated from a cell inaccordance with this invention.

Furthermore, this invention provides a nucleic acid probe comprising atleast 15 nucleotides, which probe specifically hybridizes with a nucleicacid encoding a mammalian SNORF25 receptor, wherein the probe has asequence complementary to a unique sequence present within one of thetwo strands of the nucleic acid encoding the mammalian SNORF25 receptorcontained in plasmid pEXJT3T7-hSNORF25 (ATCC Accession No. 203495) orplasmid pcDNA3.1-rSNORF25 (ATCC Accession No. 203494).

This invention further provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a mammalian SNORF25 receptor, wherein the probe has a sequencecomplementary to a unique sequence present within (a) the nucleic acidsequence shown in FIGS. 1A-1B (SEQ ID NO: 1) or (b) the reversecomplement thereof. This invention also provides a nucleic acid probecomprising at least 15 nucleotides, which probe specifically hybridizeswith a nucleic acid encoding a mammalian SNORF25 receptor, wherein theprobe has a sequence complementary to a unique sequence present within(a) the nucleic acid sequence shown in FIGS. 3A-3B (SEQ ID NO: 3) or (b)the reverse complement thereof. In one embodiment, the nucleic acid isDNA. In another embodiment, the nucleic acid is RNA.

As used herein, the phrase “specifically hybridizing” means the abilityof a nucleic acid molecule to recognize a nucleic acid sequencecomplementary to its own and to form double-helical segments throughhydrogen bonding between complementary base pairs.

The nucleic acids according to this invention may be used as probes toobtain homologous nucleic acids from other species and to detect theexistence of nucleic acids having complementary sequences in samples.

The nucleic acids may also be used to express the receptors they encodein transfected cells.

The use of a constitutively active receptor encoded by SNORF25 eitheroccurring naturally without further modification or after appropriatepoint mutations, deletions or the like, allows screening for antagonistsand in vivo use of such antagonists to attribute a role to receptorSNORF25 without prior knowledge of the endogenous ligand.

Use of the nucleic acids further enables elucidation of possiblereceptor diversity and of the existence of multiple subtypes within afamily of receptors of which SNORF25 is a member.

Finally, it is contemplated that this receptor will serve as a valuabletool for designing drugs for treating various pathophysiologicalconditions such as chronic and acute inflammation, arthritis, autoimmunediseases, transplant rejection, graft vs. host disease, bacterial,fungal, protozoan and viral infections, septicemia, AIDS, pain,psychotic and neurological disorders, including anxiety, depression,schizophrenia, dementia, mental retardation, memory loss, epilepsy,neurological disorders, neuromotor disorders, respiratory disorders,asthma, eating/body weight disorders including obesity, bulimia,diabetes, anorexia, nausea, hypertension, hypotension, vascular andcardiovascular disorders, ischemia, stroke, cancers, ulcers, urinaryretention, sexual/reproductive disorders, circadian rhythm disorders,renal disorders, bone diseases including osteoporosis, benign prostatichypertrophy, gastrointestinal disorders, nasal congestion,dermatological disorders such as psoriasis, allergies, Parkinson'sdisease, Alzheimer's disease, acute heart failure, angina disorders,delirium, dyskinesias such as Huntington's disease or Gille's de laTourette's syndrome, among others and diagnostic assays for suchconditions. This receptor may also serve as a valuable tool fordesigning drugs for chemoprevention.

Methods of transfecting cells e.g. mammalian cells, with such nucleicacid to obtain cells in which the receptor is expressed on the surfaceof the cell are well known in the art. (See, for example, U.S. Pat. Nos.5,053,337; 5,155,218; 5,360,735; 5,472,866; 5,476,782; 5,516,653;5,545,549; 5,556,753; 5,595,880; 5,602,024; 5,639,652; 5,652,113;5,661,024; 5,766,879; 5,786,155; and 5,786,157, the disclosures of whichare hereby incorporated by reference in their entireties into thisapplication.)

Such transfected cells may also be used to test compounds and screencompound libraries to obtain compounds which bind to the SNORF25receptor, as well as compounds which activate or inhibit activation offunctional responses in such cells, and therefore are likely to do so invivo. (See, for example, U.S. Pat. Nos. 5,053,337; 5,155,218; 5,360,735;5,472,866; 5,476,782; 5,516,653; 5,545,549; 5,556,753; 5,595,880;5,602,024; 5,639,652; 5,652,113; 5,661,024; 5,766,879; 5,786,155; and5,786,157, the disclosures of which are hereby incorporated by referencein their entireties into this application.)

This invention further provides an antibody capable of binding to amammalian receptor encoded by a nucleic acid encoding a mammalianreceptor. In one embodiment, the mammalian receptor is a human receptor.In a further embodiment, the mammalian receptor is a rat receptor. Thisinvention also provides an agent capable of competitively inhibiting thebinding of an antibody to a mammalian receptor. In one embodiment, theantibody is a monoclonal antibody or antisera.

Methods of preparing and employing antisense oligonucleotides,antibodies, nucleic acid probes and transgenic animals directed to theSNORF25 receptor are well known in the art. (See, for example, U.S. Pat.Nos. 5,053,337; 5,155,218; 5,360,735; 5,472,866; 5,476,782; 5,516,653;5,545,549; 5,556,753; 5,595,880; 5,602,024; 5,639,652; 5,652,113;5,661,024; 5,766,879; 5,786,155; and 5,786,157, the disclosures of whichare hereby incorporated by reference in their entireties into thisapplication.)

This invention also provides an antisense oligonucleotide having asequence capable of specifically hybridizing to RNA encoding a mammalianSNORF25 receptor, so as to prevent translation of such RNA. Thisinvention further provides an antisense oligonucleotide having asequence capable of specifically hybridizing to genomic DNA encoding amammalian SNORF25 receptor, so as to prevent transcription of suchgenomic DNA. In one embodiment, the oligonucleotide comprises chemicallymodified nucleotides or nucleotide analogues.

This invention provides an antibody capable of binding to a mammalianSNORF25 receptor encoded by a nucleic acid in accordance with thisinvention. In one embodiment, the mammalian SNORF25 receptor is a humanSNORF25 receptor. In a further embodiment, the mammalian SNORF25receptor is a rat SNORF25 receptor.

Moreover, this invention provides an agent capable of competitivelyinhibiting the binding of an antibody in accordance with this inventionto a mammalian SNORF25 receptor. In one embodiment, the antibody is amonoclonal antibody or antisera.

This invention still further provides a pharmaceutical compositioncomprising (a) an amount of an oligonucleotide in accordance with thisinvention capable of passing through a cell membrane and effective toreduce expression of a mammalian SNORF25 receptor and (b) apharmaceutically acceptable carrier capable of passing through the cellmembrane.

In one embodiment, the oligonucleotide is coupled to a substance whichinactivates mRNA. In another embodiment, the substance which inactivatesmRNA is a ribozyme. In another embodiment, the pharmaceuticallyacceptable carrier comprises a structure which binds to a mammalianSNORF25 receptor on a cell capable of being taken up by the cells afterbinding to the structure. In another embodiment, the pharmaceuticallyacceptable carrier is capable of binding to a mammalian SNORF25 receptorwhich is specific for a selected cell type.

This invention also provides a pharmaceutical composition whichcomprises an amount of an antibody in accordance with this inventioneffective to block binding of a ligand to a human SNORF25 receptor and apharmaceutically acceptable carrier.

This invention further provides a transgenic, nonhuman mammal expressingDNA encoding a mammalian SNORF25 receptor in accordance with thisinvention. This invention provides a transgenic, nonhuman mammalcomprising a homologous recombination knockout of a native mammalianSNORF25 receptor. This invention further provides a transgenic, nonhumanmammal whose genome comprises antisense DNA complementary to DNAencoding a mammalian SNORF25 receptor in accordance with this inventionso placed within such genome as to be transcribed into antisense mRNAwhich is complementary to and hybridizes with mRNA encoding themammalian SNORF25 receptor so as to reduce translation of such mRNA andexpression of such receptor. In one embodiment, the DNA encoding themammalian SNORF25 receptor additionally comprises an inducible promoter.In another embodiment, the DNA encoding the mammalian SNORF25 receptoradditionally comprises tissue specific regulatory elements. In anotherembodiment, the transgenic, nonhuman mammal is a mouse.

This invention provides a process for identifying a chemical compoundwhich specifically binds to a mammalian SNORF25 receptor which comprisescontacting cells containing DNA encoding, and expressing on their cellsurface, the mammalian SNORF25 receptor, wherein such cells do notnormally express the mammalian SNORF2S receptor, with the compound underconditions suitable for binding, and detecting specific binding of thechemical compound to the mammalian SNORF25 receptor. This inventionfurther provides a process for identifying a chemical compound whichspecifically binds to a mammalian SNORF25 receptor which comprisescontacting a membrane preparation from cells containing DNA encoding,and expressing on their cell surface, the mammalian SNORF25 receptor,wherein such cells do not normally express the mammalian SNORF25receptor, with the compound under conditions suitable for binding, anddetecting specific binding of the chemical compound to the mammalianSNORF25 receptor.

In one embodiment, the mammalian SNORF25 receptor is a human SNORF25receptor. In another embodiment, the mammalian SNORF25 receptor hassubstantially the same amino acid sequence as the human SNORF25 receptorencoded by plasmid pEXJT3T7-hSNORF25 (ATCC Accession No. 203495). Inanother embodiment, the mammalian SNORF25 receptor has substantially thesame amino acid sequence as that shown in FIGS. 2A-2B (SEQ ID NO: 2). Inanother embodiment, the mammalian SNORF25 receptor has the amino acidsequence shown in FIGS. 2A-2B (SEQ ID NO: 2).

In another embodiment, the mammalian SNORF25 receptor is a rat SNORF25receptor. In another embodiment, the mammalian SNORF25 receptor hassubstantially the same amino acid sequence as the rat SNORF25 receptorencoded by plasmid pcDNA3.1-rSNORF25 (ATCC Accession No. 203494). Inanother embodiment, the mammalian SNORF25 receptor has substantially thesame amino acid sequence as that shown in FIGS. 4A-4B (SEQ ID NO: 4). Inanother embodiment, the mammalian SNORF25 receptor has the amino acidsequence shown in FIGS. 4A-4B (SEQ ID NO: 4).

In one embodiment, the compound is not previously known to bind to amammalian SNORF25 receptor. In one embodiment, the cell is an insectcell. In one embodiment, the cell is a mammalian cell. In anotherembodiment, the cell is nonneuronal in origin. In another embodiment,the nonneuronal cell is a COS-7 cell, 293 human embryonic kidney cell, aCHO cell, a NIH-3T3 cell, a mouse Y1 cell, or a LM(tk−) cell. In anotherembodiment, the compound is a compound not previously known to bind to amammalian SNORF25 receptor. This invention provides a compoundidentified by the preceding process of this invention.

This invention still further provides a process involving competitivebinding for identifying a chemical compound which specifically binds toa mammalian SNORF25 receptor which comprises separately contacting cellsexpressing on their cell surface the mammalian SNORF25 receptor, whereinsuch cells do not normally express the mammalian SNORF25 receptor, withboth the chemical compound and a second chemical compound known to bindto the receptor, and with only the second chemical compound, underconditions suitable for binding of such compounds to the receptor, anddetecting specific binding of the chemical compound to the mammalianSNORF25 receptor, a decrease in the binding of the second chemicalcompound to the mammalian SNORF25 receptor in the presence of thechemical compound being tested indicating that such chemical compoundbinds to the mammalian SNORF25 receptor.

This invention provides a process involving competitive binding foridentifying a chemical compound which specifically binds to a mammalianSNORF25 receptor which comprises separately contacting a membranepreparation from cells expressing on their cell surface the mammalianSNORF25 receptor, wherein such cells do not normally express themammalian SNORF25 receptor, with both the chemical compound and a secondchemical compound known to bind to the receptor, and with only thesecond chemical compound, under conditions suitable for binding of suchcompounds to the receptor, and detecting specific binding of thechemical compound to the mammalian SNORF25 receptor, a decrease in thebinding of the second chemical compound to the mammalian SNORF25receptor in the presence of the chemical compound being testedindicating that such chemical compound binds to the mammalian SNORF25receptor.

In one embodiment, the mammalian SNORF25 receptor is a human SNORF25receptor. In another embodiment, the mammalian SNORF25 receptor is a ratSNORF25 receptor. In a further embodiment, the cell is an insect cell.In another embodiment, the cell is a mammalian cell. In anotherembodiment, the cell is nonneuronal in origin. In another embodiment,the nonneuronal cell is a COS-7 cell, 293 human embryonic kidney cell, aCHO cell, a NIH-3T3 cell, a mouse Y1 cell, or a LM(tk−) cell. In anotherembodiment, the compound is not previously known to bind to a mammalianSNORF25 receptor. This invention provides a compound identified by thepreceding process of this invention.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a mammalian SNORF25 receptor to identifya compound which specifically binds to the mammalian SNORF25 receptor,which comprises (a) contacting cells transfected with, and expressing,DNA encoding the mammalian SNORF25 receptor with a compound known tobind specifically to the mammalian SNORF25 receptor; (b) contacting thecells of step (a) with the plurality of compounds not known to bindspecifically to the mammalian SNORF25 receptor, under conditionspermitting binding of compounds known to bind to the mammalian SNORF25receptor; (c) determining whether the binding of the compound known tobind to the mammalian SNORF25 receptor is reduced in the presence of theplurality of compounds, relative to the binding of the compound in theabsence of the plurality of compounds; and if so (d) separatelydetermining the binding to the mammalian SNORF25 receptor of eachcompound included in the plurality of compounds, so as to therebyidentify any compound included therein which specifically binds to themammalian SNORF25 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a mammalian SNORF25 receptor to identifya compound which specifically binds to the mammalian SNORF25 receptor,which comprises (a) contacting a membrane preparation from cellstransfected with, and expressing, DNA encoding the mammalian SNORF25receptor with the plurality of compounds not known to bind specificallyto the mammalian SNORF25 receptor under conditions permitting binding ofcompounds known to bind to the mammalian SNORF25 receptor; (b)determining whether the binding of a compound known to bind to themammalian SNORF25 receptor is reduced in the presence of the pluralityof compounds, relative to the binding of the compound in the absence ofthe plurality of compounds; and if so (c) separately determining thebinding to the mammalian SNORF25 receptor of each compound included inthe plurality of compounds, so as to thereby identify any compoundincluded therein which specifically binds to the mammalian SNORF25receptor.

In one embodiment, the mammalian SNORF25 receptor is a human SNORF25receptor. In a further embodiment, the mammalian SNORF25 receptor is arat SNORF25 receptor. In another embodiment, the cell is a mammaliancell. In another embodiment, the mammalian cell is non-neuronal inorigin. In a further embodiment, the non-neuronal cell is a COS-7 cell,a 293 human embryonic kidney cell, a LM(tk−) cell, a CHO cell, a mouseY1 cell, or an NIH-3T3 cell.

This invention provides a method of detecting expression of a mammalianSNORF25 receptor by detecting the presence of mRNA coding for themammalian SNORF25 receptor which comprises obtaining total mRNA from thecell and contacting the mRNA so obtained with a nucleic acid probeaccording to this invention under hybridizing conditions, detecting thepresence of mRNA hybridized to the probe, and thereby detecting theexpression of the mammalian SNORF25 receptor by the cell.

This invention provides a method of detecting the presence of amammalian SNORF25 receptor on the surface of a cell which comprisescontacting the cell with an antibody according to this invention underconditions permitting binding of the antibody to the receptor, detectingthe presence of the antibody bound to the cell, and thereby detectingthe presence of the mammalian SNORF25 receptor on the surface of thecell.

This invention provides a method of determining the physiologicaleffects of varying levels of activity of mammalian SNORF25 receptorswhich comprises producing a transgenic, nonhuman mammal in accordancewith this invention whose levels of mammalian SNORF25 receptor activityare varied by use of an inducible promoter which regulates mammalianSNORF25 receptor expression.

This invention provides a method of determining the physiologicaleffects of varying levels of activity of mammalian SNORF25 receptorswhich comprises producing a panel of transgenic, nonhuman mammals inaccordance with this invention each expressing a different amount ofmammalian SNORF25 receptor.

This invention provides a method for identifying an antagonist capableof alleviating an abnormality wherein the abnormality is alleviated bydecreasing the activity of a mammalian SNORF25 receptor comprisingadministering a compound to a transgenic, nonhuman mammal according tothis invention, and determining whether the compound alleviates anyphysiological and/or behavioral abnormality displayed by the transgenic,nonhuman mammal as a result of overactivity of a mammalian SNORF25receptor, the alleviation of such abnormality identifying the compoundas an antagonist. In one embodiment, the mammalian SNORF25 receptor is ahuman SNORF25 receptor. In a further embodiment, the mammalian SNORF25receptor is a rat SNORF25 receptor. The invention provides an antagonistidentified by the preceding method according to this invention. Thisinvention provides a composition, e.g. a pharmaceutical composition,comprising an antagonist according to this invention and a carrier, e.g.a pharmaceutically acceptable carrier. This invention provides a methodof treating an abnormality in a subject wherein the abnormality isalleviated by decreasing the activity of a mammalian SNORF25 receptorwhich comprises administering to the subject an effective amount of thepharmaceutical composition according to this invention so as to therebytreat the abnormality.

This invention provides a method for identifying an agonist capable ofalleviating an abnormality in a subject wherein the abnormality isalleviated by increasing the activity of a mammalian SNORF25 receptorcomprising administering a compound to a transgenic, nonhuman mammalaccording to this invention, and determining whether the compoundalleviates any physiological and/or behavioral abnormality displayed bythe transgenic, nonhuman mammal, the alleviation of such an abnormalityidentifying the compound as an agonist. In one embodiment, the mammalianSNORF25 receptor is a human SNORF25 receptor. In a further embodiment,the mammalian SNORF25 receptor is a rat SNORF25 receptor. This inventionprovides an agonist identified by the preceding method according to thisinvention. This invention provides a composition, e.g. a pharmaceuticalcomposition, comprising an agonist identified by the method according tothis invention and a carrier, e.g. a pharmaceutically acceptablecarrier.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by increasing the activity of amammalian SNORF25 receptor which comprises administering to the subjectan effective amount of the pharmaceutical composition according to thisinvention so as to thereby treat the abnormality.

This invention provides a method for diagnosing a predisposition to adisorder associated with the activity of a specific mammalian allelewhich comprises: (a) obtaining DNA of subjects suffering from thedisorder; (b) performing a restriction digest of the DNA with a panel ofrestriction enzymes; (c) electrophoretically separating the resultingDNA fragments on a sizing gel; (d) contacting the resulting gel with anucleic acid probe capable of specifically hybridizing with a uniquesequence included within the sequence of a nucleic acid moleculeencoding a mammalian SNORF25 receptor and labeled with a detectablemarker; (e) detecting labeled bands which have hybridized to the DNAencoding a mammalian SNORF25 receptor to create a unique band patternspecific to the DNA of subjects suffering from the disorder; (f)repeating steps (a)-(e) with DNA obtained for diagnosis from subjectsnot yet suffering from the disorder; and (g) comparing the unique bandpattern specific to the DNA of subjects suffering from the disorder fromstep (e) with the band pattern from step (f) for subjects not yetsuffering from the disorder so as to determine whether the patterns arethe same or different and thereby diagnose predisposition to thedisorder if the patterns are the same.

In one embodiment, the disorder is a disorder associated with theactivity of a specific mammalian allele is diagnosed.

This invention provides a method of preparing a purified mammalianSNORF25 receptor according to this invention which comprises: (a)culturing cells which express the mammalian SNORF25 receptor; (b)recovering the mammalian SNORF25 receptor from the cells; and (c)purifying the mammalian SNORF25 receptor so recovered.

This invention provides a method of preparing the purified mammalianSNORF25 receptor according to this invention which comprises: (a)inserting a nucleic acid encoding the mammalian SNORF25 receptor into asuitable expression vector; (b) introducing the resulting vector into asuitable host cell; (c) placing the resulting host cell in suitableconditions permitting the production of the mammalian SNORF25 receptor;(d) recovering the mammalian SNORF25 receptor so produced; andoptionally (e) isolating and/or purifying the mammalian SNORF25 receptorso recovered.

This invention provides a process for determining whether a chemicalcompound is a mammalian SNORF25 receptor agonist which comprisescontacting cells transfected with and expressing DNA encoding themammalian SNORF25 receptor with the compound under conditions permittingthe activation of the mammalian SNORF25 receptor, and detecting anyincrease in mammalian SNORF25 receptor activity, so as to therebydetermine whether the compound is a mammalian SNORF25 receptor agonist.

This invention provides a process for determining whether a chemicalcompound is a mammalian SNORF25 receptor antagonist which comprisescontacting cells transfected with and expressing DNA encoding themammalian SNORF25 receptor with the compound in the presence of a knownmammalian SNORF25 receptor agonist, under conditions permitting theactivation of the mammalian SNORF25 receptor, and detecting any decreasein mammalian SNORF25 receptor activity, so as to thereby determinewhether the compound is a mammalian SNORF25 receptor antagonist.

In one embodiment, the mammalian SNORF25 receptor is a human SNORF25receptor. In another embodiment, the mammalian SNORF25 receptor is a ratSNORF25 receptor.

This invention provides a composition, for example a pharmaceuticalcomposition, which comprises an amount of a mammalian SNORF25 receptoragonist determined by a process according to this invention effective toincrease activity of a mammalian SNORF25 receptor and a carrier, forexample, a pharmaceutically acceptable carrier. In one embodiment, themammalian SNORF25 receptor agonist is not previously known.

This invention provides a composition, for example a pharmaceuticalcomposition, which comprises an amount of a mammalian SNORF25 receptorantagonist determined by a process according to this invention effectiveto reduce activity of a mammalian SNORF25 receptor and a carrier, forexample, a pharmaceutically acceptable carrier. In one embodiment, themammalian SNORF25 receptor antagonist is not previously known.

This invention provides a process for determining whether a chemicalcompound specifically binds to and activates a mammalian SNORF25receptor, which comprises contacting cells producing a second messengerresponse and expressing on their cell surface the mammalian SNORF25receptor, wherein such cells do not normally express the mammalianSNORF25 receptor, with the chemical compound under conditions suitablefor activation of the mammalian SNORF25 receptor, and measuring thesecond messenger response in the presence and in the absence of thechemical compound, a change, e.g. an increase, in the second messengerresponse in the presence of the chemical compound indicating that thecompound activates the mammalian SNORF25 receptor.

In one embodiment, the second messenger response comprises chloridechannel activation and the change in second messenger is an increase inthe level of chloride current. In another embodiment, the secondmessenger response comprises change in intracellular calcium levels andthe change in second messenger is an increase in the measure ofintracellular calcium. In another embodiment, the second messengerresponse comprises release of inositol phosphate and the change insecond messenger is an increase in the level of inositol phosphate. Inanother embodiment, the second messenger response comprises release ofarachidonic acid and the change in second messenger is an increase inthe level of arachidonic acid. In yet another embodiment, the secondmessenger response comprises GTPγS ligand binding and the change insecond messenger is an increase in GTPγS ligand binding. In anotherembodiment, the second messenger response comprises activation of MAPkinase and the change in second messenger response is an increase in MAPkinase activation. In a further embodiment, the second messengerresponse comprises cAMP accumulation and the change in second messengerresponse is an increase in cAMP accumulation.

This invention provides a process for determining whether a chemicalcompound specifically binds to and inhibits activation of a mammalianSNORF25 receptor, which comprises separately contacting cells producinga second messenger response and expressing on their cell surface themammalian SNORF25 receptor, wherein such cells do not normally expressthe mammalian SNORF25 receptor, with both the chemical compound and asecond chemical compound known to activate the mammalian SNORF25receptor, and with only the second chemical compound, under conditionssuitable for activation of the mammalian SNORF25 receptor, and measuringthe second messenger response in the presence of only the secondchemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change, e.g. increase, inthe second messenger response in the presence of both the chemicalcompound and the second chemical compound than in the presence of onlythe second chemical compound indicating that the chemical compoundinhibits activation of the mammalian SNORF25 receptor.

In one embodiment, the second messenger response comprises chloridechannel activation and the change in second messenger response is asmaller increase in the level of chloride current in the presence ofboth the chemical compound and the second chemical compound than in thepresence of only the second chemical compound. In another embodiment,the second messenger response comprises change in intracellular calciumlevels and the change in second messenger response is a smaller increasein the measure of intracellular calcium in the presence of both thechemical compound and the second chemical compound than in the presenceof only the second chemical compound. In another embodiment, the secondmessenger response comprises release of inositol phosphate and thechange in second messenger response is a smaller increase in the levelof inositol phosphate in the presence of both the chemical compound andthe second chemical compound than in the presence of only the secondchemical compound.

In one embodiment, the second messenger response comprises activation ofMAP kinase and the change in second messenger response is a smallerincrease in the level of MAP kinase activation in the presence of boththe chemical compound and the second chemical compound than in thepresence of only the second chemical compound. In another embodiment,the second messenger response comprises change in cAMP levels and thechange in second messenger response is a smaller change in the level ofcAMP in the presence of both the chemical compound and the secondchemical compound than in the presence of only the second chemicalcompound. In another embodiment, the second messenger response comprisesrelease of arachidonic acid and the change in second messenger responseis an increase in the level of arachidonic acid levels in the presenceof both the chemical compound and the second chemical compound than inthe presence of only the second chemical compound. In a furtherembodiment, the second messenger response comprises GTPγS ligand bindingand the change in second messenger is a smaller increase in GTPγS ligandbinding in the presence of both the chemical compound and the secondchemical compound than in the presence of only the second chemicalcompound.

In one embodiment, the mammalian SNORF25 receptor is a human SNORF25receptor. In a further embodiment, the mammalian SNORF25 receptor is arat SNORF25 receptor. In another embodiment, the cell is an insect cell.In another embodiment, the cell is a mammalian cell. In anotherembodiment, the mammalian cell is nonneuronal in origin. In anotherembodiment, the nonneuronal cell is a COS-7 cell, CHO cell, 293 humanembryonic kidney cell, NIH-3T3 cell or LM(tk−) cell. In anotherembodiment, the compound is not previously known to bind to a mammalianSNORF25 receptor.

This invention provides a compound determined by a process according tothis invention and a composition, for example, a pharmaceuticalcomposition, which comprises an amount of a mammalian SNORF25 receptoragonist determined to be such by a process according to this inventioneffective to increase activity of the mammalian SNORF25 receptor and acarrier, for example, a pharmaceutically acceptable carrier. In oneembodiment, the mammalian SNORF25 receptor agonist is not previouslyknown.

This invention provides a composition, for example, a pharmaceuticalcomposition, which comprises an amount of a mammalian SNORF25 receptorantagonist determined to be such by a process according to thisinvention, effective to reduce activity of the mammalian SNORF25receptor and a carrier, for example, a pharmaceutically acceptablecarrier. In one embodiment, the mammalian SNORF25 receptor antagonist isnot previously known.

This invention provides a method of screening a plurality of chemicalcompounds not known to activate a mammalian SNORF25 receptor to identifya compound which activates the mammalian SNORF25 receptor whichcomprises: (a) contacting cells transfected with and expressing themammalian SNORF25 receptor with the plurality of compounds not known toactivate the mammalian SNORF25 receptor, under conditions permittingactivation of the mammalian SNORF25 receptor; (b) determining whetherthe activity of the mammalian SNORF25 receptor is increased in thepresence of one or more of the compounds; and if so (c) separatelydetermining whether the activation of the mammalian SNORF25 receptor isincreased by any compound included in the plurality of compounds, so asto thereby identify each compound which activates the mammalian SNORF25receptor. In one embodiment, the mammalian SNORF25 receptor is a humanSNORF25 receptor. In a further embodiment, the mammalian SNORF25receptor is a rat SNORF25 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to inhibit the activation of a mammalian SNORF25receptor to identify a compound which inhibits the activation of themammalian SNORF25 receptor, which comprises: (a) contacting cellstransfected with and expressing the mammalian SNORF25 receptor with theplurality of compounds in the presence of a known mammalian SNORF25receptor agonist, under conditions permitting activation of themammalian SNORF25 receptor; (b) determining whether the extent or amountof activation of the mammalian SNORF25 receptor is reduced in thepresence of one or more of the compounds, relative to the extent oramount of activation of the mammalian SNORF25 receptor in the absence ofsuch one or more compounds; and if so (c) separately determining whethereach such compound inhibits activation of the mammalian SNORF25 receptorfor each compound included in the plurality of compounds, so as tothereby identify any compound included in such plurality of compoundswhich inhibits the activation of the mammalian SNORF25 receptor.

In one embodiment, the mammalian SNORF25 receptor is a human SNORF25receptor. In a further embodiment, the mammalian SNORF25 receptor is arat SNORF25 receptor. In another embodiment, wherein the cell is amammalian cell. In another embodiment, the mammalian cell isnon-neuronal in origin. In another embodiment, the non-neuronal cell isa COS-7 cell, a 293 human embryonic kidney cell, a LM(tk−) cell or anNIH-3T3 cell.

This invention provides a composition, for example a pharmaceuticalcomposition, comprising a compound identified by a method according tothis invention in an amount effective to increase mammalian SNORF25receptor activity and a carrier, for example, a pharmaceuticallyacceptable carrier.

This invention provides a composition, for example, a pharmaceuticalcomposition, comprising a compound identified by a method according tothis invention in an amount effective to decrease mammalian SNORF25receptor activity and a carrier, for example, a pharmaceuticallyacceptable carrier.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by increasing the activity of amammalian SNORF25 receptor which comprises administering to the subjecta compound which is a mammalian SNORF25 receptor agonist in an amounteffective to treat the abnormality. In one embodiment, the abnormalityis a regulation of a steroid hormone disorder, an epinephrine releasedisorder, a gastrointestinal disorder, a cardiovascular disorder, anelectrolyte balance disorder, hypertension, diabetes, a respiratorydisorder, asthma, a reproductive function disorder, an immune disorder,an endocrine disorder, a musculoskeletal disorder, a neuroendocrinedisorder, a cognitive disorder, a memory disorder, somatosensory andneurotransmission disorders, a motor coordination disorder, a sensoryintegration disorder, a motor integration disorder, a dopaminergicfunction disorder, an appetite disorder, such as anorexia or obesity, asensory transmission disorder, an olfaction disorder, an autonomicnervous system disorder, pain, psychotic behavior, affective disorder,migraine, cancer, proliferative diseases, wound healing, tissueregeneration, blood coagulation-related disorders, developmentaldisorders, or ischemia-reperfusion injury-related diseases.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by decreasing the activity of amammalian SNORF25 receptor which comprises administering to the subjecta compound which is a mammalian SNORF25 receptor antagonist in an amounteffective to treat the abnormality. In one embodiment, the abnormalityis a regulation of a steroid hormone disorder, an epinephrine releasedisorder, a gastrointestinal disorder, a cardiovascular disorder, anelectrolyte balance disorder, hypertension, diabetes, a respiratorydisorder, asthma, a reproductive function disorder, an immune disorder,an endocrine disorder, a musculoskeletal disorder, a neuroendocrinedisorder, a cognitive disorder, a memory disorder, somatosensory andneurotransmission disorders, a motor coordination disorder, a sensoryintegration disorder, a motor integration disorder, a dopaminergicfunction disorder, an appetite disorder, such as anorexia or obesity, asomatosensory neurotransmission disorder, an olfaction disorder, anautonomic nervous system disorder, pain, psychotic behavior, affectivedisorder, migraine, cancer, proliferative diseases, wound healing,tissue regeneration, blood coagulation-related disorders, developmentaldisorders, or ischemia-reperfusion injury-related diseases.

This invention provides a process for making a composition of matterwhich specifically binds to a mammalian SNORF25 receptor which comprisesidentifying a chemical compound using a process in accordance with thisinvention and then synthesizing the chemical compound or a novelstructural and functional analog or homolog thereof. In one embodiment,the mammalian SNORF25 receptor is a human SNORF25 receptor. In anotherembodiment, the mammalian SNORF25 receptor is a rat SNORF25 receptor.

This invention provides a process for preparing a composition, forexample, a pharmaceutical composition which comprises admixing acarrier, for example, a pharmaceutically acceptable carrier, and apharmaceutically effective amount of a chemical compound identified by aprocess in accordance with this invention or a novel structural andfunctional analog or homolog thereof. In one embodiment, the mammalianSNORF25 receptor is a human SNORF25 receptor. In another embodiment, themammalian SNORF25 receptor is a rat SNORF25 receptor.

Thus, once the gene for a targeted receptor subtype is cloned, it isplaced into a recipient cell which then expresses the targeted receptorsubtype on its surface. This cell, which expresses a single populationof the targeted human receptor subtype, is then propagated resulting inthe establishment of a cell line. This cell line, which constitutes adrug discovery system, is used in two different types of assays: bindingassays and functional assays. In binding assays, the affinity of acompound for both the receptor subtype that is the target of aparticular drug discovery program and other receptor subtypes that couldbe associated with side effects are measured. These measurements enableone to predict the potency of a compound, as well as the degree ofselectivity that the compound has for the targeted receptor subtype overother receptor subtypes. The data obtained from binding assays alsoenable chemists to design compounds toward or away from one or more ofthe relevant subtypes, as appropriate, for optimal therapeutic efficacy.In functional assays, the nature of the response of the receptor subtypeto the compound is determined. Data from the functional assays showwhether the compound is acting to inhibit or enhance the activity of thereceptor subtype, thus enabling pharmacologists to evaluate compoundsrapidly at their ultimate human receptor subtypes targets permittingchemists to rationally design drugs that will be more effective and havefewer or substantially less severe side effects than existing drugs.

Approaches to designing and synthesizing receptor subtype-selectivecompounds are well known and include traditional medicinal chemistry andthe newer technology of combinatorial chemistry, both of which aresupported by computer-assisted molecular modeling. With such approaches,chemists and pharmacologists use their knowledge of the structures ofthe targeted receptor subtype and compounds determined to bind and/oractivate or inhibit activation of the receptor subtype to design andsynthesize structures that will have activity at these receptorsubtypes.

Combinatorial chemistry involves automated synthesis of a variety ofnovel compounds by assembling them using different combinations ofchemical building blocks. The use of combinatorial chemistry greatlyaccelerates the process of generating compounds. The resulting arrays ofcompounds are called libraries and are used to screen for compounds(“lead compounds”) that demonstrate a sufficient level of activity atreceptors of interest. Using combinatorial chemistry it is possible tosynthesize “focused” libraries of compounds anticipated to be highlybiased toward the receptor target of interest.

Once lead compounds are identified, whether through the use ofcombinatorial chemistry or traditional medicinal chemistry or otherwise,a variety of homologs and analogs are prepared to facilitate anunderstanding of the relationship between chemical structure andbiological or functional activity. These studies define structureactivity relationships which are then used to design drugs with improvedpotency, selectivity and pharmacokinetic properties. Combinatorialchemistry is also used to rapidly generate a variety of structures forlead optimization. Traditional medicinal chemistry, which involves thesynthesis of compounds one at a time, is also used for furtherrefinement and to generate compounds not accessible by automatedtechniques. Once such drugs are defined the production is scaled upusing standard chemical manufacturing methodologies utilized throughoutthe pharmaceutical and chemistry industry.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS Materials and Methods Mixed Oligonucleotide PrimedAmplification of cDNA (MOPAC)

Mixed Oligonucleotide Primed Amplification of cDNA (MOPAC) was performedon several DNA templates including: rat genomic DNA, cDNAreverse-transcribed from mRNA isolated from the GH1 cell line, and theRin14b cell line. The MOPAC reaction was performed using Taq DNApolymerase (Boehringer-Mannheim, Indianapolis, Ind.) and the followingdegenerate oligonucleotides: JAB55, designed based on the thirdtransmembrane domain of the galanin, somatostatin, and opiate receptorfamilies; and TL1020, designed based on the 7^(th) transmembrane domainof the galanin receptor family.

The conditions for the MOPAC PCR reaction were as follows: 3 minute holdat 94° C.; 10 cycles of 1 minute at 94° C., 1 minute 45 seconds at 44°C., 2 minutes at 72° C.; 30 cycles of 94° C. for 1 minute, 49° C. for 1minute 45 seconds, 2 minutes at 72° C.; 4 minute hold at 72° C.; 4° C.hold until ready for agarose gel electrophoresis.

The products were run on a 1% agarose TAE gel and bands of the expectedsize (˜500-600 bp) were cut from the gel, purified using the QIAQUICKgel extraction kit (QIAGEN, Chatsworth, Calif.), and subcloned into theTA cloning vector (Invitrogen, San Diego, Calif.). White(insert-containing) colonies were picked and subjected to PCR usingpCR2.1 vector primers JAB1 and JAB2 using the following protocol: 94° C.hold for 3 minutes; 35 cycles of 94° C. for 1 minute, 68° C. for 1minute 15 seconds; 2 minute hold at 68° C., 4° C. hold until theproducts were ready for purification. PCR products were purified byisopropanol precipitation (10 μl PCR product, 18 μl low TE, 10.5 μl 2MNaClO₄, and 21.5 μl isopropanol) and sequenced using the ABI Big Dyecycle sequencing protocol and ABI 377 sequencers (ABI, Foster City,Calif.). One of these PCR products, later named SNORF25, was determinedto be a novel G protein-coupled receptor-like sequence based on databasesearches and its homology to other known G protein-coupled receptors(˜29% identity to the known receptors dopamine D1, beta-adrenergic 2band 5-HT1f; 34% identity to the 5-HT4l receptor).

5′ and 3′ RACE

To determine the full-length coding sequence of SNORF25, the ClontechMarathon cDNA Amplification kit (Clontech, Palo Alto, Calif.) for 5′/3′Rapid Amplification of cDNA ends (RACE) was utilized. Total RNA fromRin14b cells was PolyA⁺-selected using a FastTrack mRNA Isolation Kit(Invitrogen). For 5′RACE, double-stranded cDNA was synthesized from 1 μgpolyA⁺ RNA using primer JAB73, a reverse primer from the putative fifthtransmembrane domain of the PCR fragment described above (SNORF25).Adaptor ligation and nested PCR were performed according to the MarathoncDNA Amplification protocol using Advantage Klentaq Polymerase(Clontech, Palo Alto, Calif.). The initial PCR was performed on a50-fold dilution of the ligated cDNA using the supplier's Adaptor Primer1 and JAB71, a reverse primer from the 5′end of the fifth transmembranedomain of the PCR fragment described above. One μl of this initial PCRreaction was re-amplified using the Adaptor Primer 2 and JAB69, areverse primer just downstream of the fourth transmembrane domain. Theconditions for PCR were 1 minute at 94° C.; 5 cycles of 94° C. for 15seconds and 72° C. for 1 minute 30 seconds; 5 cycles of 94° C. for 15seconds and 70° C. for 1 minute 30 seconds; 22 cycles of 94° C. for 15seconds and 68° C. for 1 minute 30 seconds; 68° C. hold for 5 minutes,and 4° C. hold until the products were ready for analysis. A 600 bpfragment from the nested PCR was isolated from a 1% agarose TAE gelusing the QIAQUICK kit and sequenced using ABI 377 sequencers and BigDyetermination cycle sequencing as described above. Sequences were analyzedusing the Wisconsin Package (GCG, Genetics Computer Group, Madison,Wis.).

For 3′ RACE, double stranded cDNA was synthesized from 1 μg polyA⁻ RNAusing the cDNA synthesis primer CDS supplied with the Marathon cDNAAmplification Kit (Clontech). PCR conditions for the 3′ RACE reactionswere similar to the 5′ RACE reactions, except that JAB74 and JAB72,forward primers from the sequence located between the fifth and sixthtransmembrane domains of the novel PCR fragment from MOPAC describedabove, were used in place of JAB 71 and JAB73, respectively. A 1.4 kbfragment from the nested PCR was isolated from a 1% agarose TAE gelusing the QIAQUICK gel purification kit (QIAGEN) and sequenced as above.

After determining the full-length coding sequence of this receptorsequence, the entire coding region was amplified from Rin14b cell linecDNA and rat genomic DNA using the Expand Long PCR system(Boehringer-Mannheim). The primers for this reaction were specific tothe 5′ and 3′ untranslated regions of SNORF25 with BamHI and HindIIIrestriction sites incorporated into the 5′ ends of the 5′ (JAB86) and 3′(JAB84) primers, respectively. The products from this reaction were thendigested with BamHI and HindIII, subcloned into the BamHI/HindIII siteof the expression vector pcDNA3.1 (−), and sequenced in both directionsusing vector- and gene-specific primers. Double-stranded sequence fromthe Rin14b-cloned SNORF25 product agreed with the sequence of the samegene amplified from rat genomic DNA. This receptor/expression vectorconstruct of rat SNORF25 in pcDNA3.1(−) was named pcDNA3.1-rSNORF25.

Homology cloning of the human homolog of SNORF25

To clone the human homolog of SNORF25, two oligonucleotide probes weredesigned based on the second (BB426) and fifth (BB427) transmembranedomains (TMs) of the rat SNORF25 sequence, and used to probe a humangenomic cosmid library (Clontech). Both primers were end-labeled withα³²P-dATP and terminal transferase (Promega, Madison, Wis.).Hybridization was performed under medium stringency conditions: 40° C.in a solution containing 37.5% formamide 5× SSC (1× SSC is 0.15M sodiumchloride, 0.015M sodium citrate), 1× Denhardt's solution (0.02%polyvinylpyrrolindone, 0.02% Ficoll, 0.02% bovine serum albumin), 7 mMTris, and 25 μg/ml sonicated salmon sperm DNA. The filters were washedthree times for 20 minutes at room temperature in a buffer containing 2×SSC/0.1% sodium dodecyl sulfate; two times for 20 minutes in a buffercontaining 0.1× SSC/0.1% sodium dodecyl sulfate, and exposed at −70° C.to Kodak BioMax MS film in the presence of an intensifying screen.

Cosmid clones hybridizing with the probes were picked, streaked onplates, and screened a second time with the same probes to verify andisolate the individual positive colonies under the same conditions.Cosmid DNA from positive colonies was digested with BamHI and HindIII,run on an agarose gel, transferred to nitrocellulose, and probed with³²P-labelled BB426. A fragment of approximately 1.9 kb from clone #45a(COS4 library) that hybridized to the probe was subcloned into theBamHI/HindIII site of pEXJT3T7, an Okayama and Berg expression vectormodified from pcEXV (Miller and Germain, 1986) to contain BstXI andother additional restriction sites as well as T3 and T7 promoters(Stratagene), and sequenced on both strands as described above. Theconstruct of the human SNORF25 receptor in this vector is namedpEXJT3T7-hSNORF25. Human SNORF25 was analyzed using the GCG software andwas determined to contain the full-length sequence of human SNORF25,having 80% amino acid identity and 83% nucleotide identity to the ratreceptor.

Oligonucleotide primers

The following is a list of primers and their associated sequences whichwere used in the cloning of these receptors:

JAB55: 5′-TBDSYVYIGAYMGITAYVTKG-3′ (SEQ ID NO: 5) TL1020:5′-GAIRSIARIGMRTAIAYIAKIGGRTT-3′ (SEQ ID NO: 6) JAB1:5′-TTATGCTTCCGGCTCGTATGTTGTG-3′ (SEQ ID NO: 7) JAB2:5′-ATGTGCTGCAAGGCGATTTAAGTTGGG-3′ (SEQ ID NO: 8) JAB69:5′-TGGTCTGCTGGAATATGGAG-3′ (SEQ ID NO: 9) JAB71:5′-CTTGGGTGAAACACAGCAAAGAAGG-3′ (SEQ ID NO: 10) JAB72:5′-ATGGAACATGCAGGAGCCATGGTTGG-3′ (SEQ ID NO: 11) JAB73:5′-AAGACAAAGAGGAGCACAGCTGGG-3′ (SEQ ID NO: 12) JAB74:5′-GCTCAAGATTGCCTCTGTGCACAG-3′ (SEQ ID NO: 13) JAB84:5′-ATCTATAAGCTTAGGCACTTGGAAACATCCATTCC-3′ (SEQ ID NO: 14) JAB86:5′-ATCTATGGATCCTGTGAGAATCTGAGCTCAAGACCC-3′ (SEQ ID NO: 15) BB426:5′-TTCACCTTAAATCTGGCCGTGGCTGATACCTTGAT- (SEQ ID NO: 16)   TGGCGTGGCTATTTCTGGGCTAG-3′ BB427:5′-GCTGTGTTTCACCCAAGGTTTGTGCTGACCCTCTC- (SEQ ID NO: 17)   CTGTGCTGGCTTCTTCCCAGCTGTGC-3′

Isolation of other species homologs of SNORF25 receptor cDNA

A nucleic acid sequence encoding a SNORF25 receptor cDNA from otherspecies may be isolated using standard molecular biology techniques andapproaches such as those described below:

Approach #1: A genomic library (e.g., cosmid, phage, P1, BAC, YAC)generated from the species of interest may be screened with a³²P-labeled oligonucleotide probe corresponding to a fragment of thehuman or rat SNORF25 receptors whose sequence is shown in FIGS. 1A-1Band 3A-3B to isolate a genomic clone. The full-length sequence may beobtained by sequencing this genomic clone. If one or more introns arepresent in the gene, the full-length intronless gene may be obtainedfrom cDNA using standard molecular biology techniques. For example, aforward PCR primer designed in the 5′UT and a reverse PCR primerdesigned in the 3′UT may be used to amplify a full-length, intronlessreceptor from cDNA. Standard molecular biology techniques could be usedto subclone this gene into a mammalian expression vector.

Approach #2: Standard molecular biology techniques may be used to screencommercial cDNA phage libraries of the species of interest byhybridization under reduced stringency with a ³²P-labeledoligonucleotide probe corresponding to a fragment of the sequences shownin FIGS. 1A-1B or 3A-3B. One may isolate a full-length SNORF25 receptorby obtaining a plaque purified clone from the lambda libraries and thensubjecting the clone to direct DNA sequencing. Alternatively, standardmolecular biology techniques could be used to screen cDNA plasmidlibraries by PCR amplification of library pools using primers designedagainst a partial species homolog sequence. A full-length clone may beisolated by Southern hybridization of colony lifts of positive poolswith a ³²P-oligonucleotide probe.

Approach #3: 3′ and 5′ RACE may be utilized to generate PCR productsfrom cDNA derived from the species of interest expressing SNORF25 whichcontain the additional sequence of SNORF25. These RACE PCR products maythen be sequenced to determine the additional sequence. This newsequence is then used to design a forward PCR primer in the 5′UT and areverse primer in the 3′UT. These primers are then used to amplify afull-length SNORF25 clone from cDNA.

Examples of other species include, but are not limited to, mouse, dog,monkey, hamster and guinea pig.

Host cells

A broad variety of host cells can be used to study heterologouslyexpressed proteins. These cells include but are not limited to mammaliancell lines such as; Cos-7, CHO, LM(tk⁻), HEK293, etc.; insect cell linessuch as; Sf9, Sf21, etc.; amphibian cells such as Xenopus oocytes;assorted yeast strains; assorted bacterial cell strains; and others.Culture conditions for each of these cell types is specific and is knownto those familiar with the art. The cells used to express SNORF25receptor were Cos-7 and Chinese hamster ovary (CHO) cells.

COS-7 cells are grown on 150 mm plates in DMEM with supplements(Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mMglutamine, 100 units/ml penicillin/100 μg/ml streptomycin) at 37° C., 5%CO₂. Stock plates of COS-7 cells are trypsinized and split 1:6 every 3-4days.

CHO cells are grown on 150 mm plates in HAM's F-12 medium withsupplements (10% bovine calf serum, 4 mM L-glutamine and 100 units/mlpenicillin/100 μg/ml streptomycin) at 37° C., 5% CO₂. Stock plates ofCHO cells are trypsinized and split 1:8 every 3-4 days.

Transient expression

DNA encoding proteins to be studied can be transiently expressed in avariety of mammalian, insect, amphibian, yeast, bacterial and other celllines by several methods, such as, calcium phosphate-mediated,DEAE-dextran mediated, liposomal-mediated, viral-mediated,electroporation-mediated and microinjection delivery. Each of thesemethods may require optimization of assorted experimental parametersdepending on the DNA, cell line, and the type of assay to besubsequently employed. The electroporation method was used totransiently transfect various cell lines with SNORF25 cDNA.

A typical protocol for the electroporation method as applied to Cos-7cells is described as follows. Cells to be used for transfection aresplit 24 hours prior to the transfection to provide flasks which aresubconfluent at the time of transfection. The cells are harvested bytrypsinization resuspended in their growth media and counted. 5×10⁶cells are suspended in 300 μl of DMEM and placed into an electroporationcuvette. 8 μg of receptor DNA plus 8 μg of any additional DNA needed(e.g. G protein expression vector, reporter construct, antibioticresistance marker, mock vector, etc.) is added to the cell suspension,the cuvette is placed into a BioRad Gene Pulser and subjected to anelectrical pulse (Gene Pulser settings: 0.25 kV voltage, 950 μFcapacitance). Following the pulse, 800 μl of complete DMEM is added toeach cuvette and the suspension transferred to a sterile tube. Completemedium is added to each tube to bring the final cell concentration to1×10⁵ cells/100 μl. The cells are then plated as needed depending uponthe type of assay to be performed.

Stable expression

Heterologous DNA can be stably incorporated into host cells, causing thecell to perpetually express a foreign protein. Methods for the deliveryof the DNA into the cell are similar to those described above fortransient expression but require the co-transfection of an ancillarygene to confer drug resistance on the targeted host cell. The ensuingdrug resistance can be exploited to select and maintain cells that havetaken up the DNA. An assortment of resistance genes are availableincluding but not restricted to neomycin, kanamycin, and hygromycin. Forthe purposes of studies concerning the receptor of this invention,stable expression of a heterologous receptor protein is typicallycarried out in, mammalian cells including but not necessarily restrictedto, CHO, HEK293, LM(tk−), etc.

In addition native cell lines that naturally carry and express thenucleic acid sequences for the receptor may be used without the need toengineer the receptor complement.

Membrane preparations

Cell membranes expressing the receptor protein according to thisinvention are useful for certain types of assays including but notrestricted to ligand binding assays, GTP-γ-S binding assays, and others.The specifics of preparing such cell membranes may in some cases bedetermined by the nature of the ensuing assay but typically involveharvesting whole cells and disrupting the cell pellet by sonication inice cold buffer (e.g. 20 mM Tris-HCl, 5 mM EDTA, pH 7.4). The resultingcrude cell lysate is cleared of cell debris by low speed centrifugationat 200×g for 5 min at 4° C. The cleared supernatant is then centrifugedat 40,000×g for 20 min at 4° C., and the resulting membrane pellet iswashed by suspending in ice cold buffer and repeating the high speedcentrifugation step. The final washed membrane pellet is resuspended inassay buffer. Protein concentrations are determined by the method ofBradford (1976) using bovine serum albumin as a standard. The membranesmay be used immediately or frozen for later use.

Generation of baculovirus

The coding region of DNA encoding the human receptor disclosed hereinmay be subcloned into pBlueBacIII into existing restriction sites orsites engineered into sequences 5′ and 3′ to the coding region of thepolypeptides. To generate baculovirus, 0.5 μg of viral DNA (BaculoGold)and 3 μg of DNA construct encoding a polypeptide may be co-transfectedinto 2×10⁶ Spodoptera frugiperda insect Sf9 cells by the calciumphosphate co-precipitation method, as outlined by Pharmingen (in“Baculovirus Expression Vector System: Procedures and Methods Manual”).The cells then are incubated for 5 days at 27° C.

The supernatant of the co-transfection plate may be collected bycentrifugation and the recombinant virus plaque purified. The procedureto infect cells with virus, to prepare stocks of virus and to titer thevirus stocks are as described in Pharmingen's manual.

Labeled ligand binding assays

Cells expressing the receptor according to this invention may be used toscreen for ligands for said receptors, for example, by labeled ligandbinding assays. Once a ligand is identified the same assays may be usedto identify agonists or antagonists of the receptor that may be employedfor a variety of therapeutic purposes.

In an embodiment, labeled ligands are placed in contact with eithermembrane preparations or intact cells expressing the receptor inmulti-well microtiter plates, together with unlabeled compounds, andbinding buffer. Binding reaction mixtures are incubated for times andtemperatures determined to be optimal in separate equilibrium bindingassays. The reaction is stopped by filtration through GF/B filters,using a cell harvester, or by directly measuring the bound ligand. Ifthe ligand was labeled with a radioactive isotope such as ³H, ¹⁴C, 125I,³⁵S, ³²P, ³³P, etc., the bound ligand may be detected by using liquidscintillation counting, scintillation proximity, or any other method ofdetection for radioactive isotopes. If the ligand was labeled with afluorescent compound, the bound labeled ligand may be measured bymethods such as, but not restricted to, fluorescence intensity, timeresolved fluorescence, fluorescence polarization, fluorescence transfer,or fluorescence correlation spectroscopy. In this manner agonist orantagonist compounds that bind to the receptor may be identified as theyinhibit the binding of the labeled ligand to the membrane protein orintact cells expressing the receptor. Non-specific binding is defined asthe amount of labeled ligand remaining after incubation of membraneprotein in the presence of a high concentration (e.g., 100-1000×K_(D))of unlabeled ligand. In equilibrium saturation binding assays membranepreparations or intact cells transfected with the receptor are incubatedin the presence of increasing concentrations of the labeled compound todetermine the binding affinity of the labeled ligand. The bindingaffinities of unlabeled compounds may be determined in equilibriumcompetition binding assays, using a fixed concentration of labeledcompound in the presence of varying concentrations of the displacingligands.

Functional assays

Cells expressing the SNORF25 receptor DNA may be used to screen forligands to SNORF25 receptor using functional assays. Once a ligand isidentified the same assays may be used to identify agonists orantagonists of the SNORF25 receptor that may be employed for a varietyof therapeutic purposes. It is well known to those in the art that theover-expression of a GPCR can result in the constitutive activation ofintracellular signaling pathways. In the same manner, over-expression ofthe SNORF25 receptor in any cell line as described above, can result inthe activation of the functional responses described below, and any ofthe assays herein described can be used to screen for both agonist andantagonist ligands of the SNORF25 receptor.

A wide spectrum of assays can be employed to screen for the presence ofSNORF25 receptor ligands. These assays range from traditionalmeasurements of total inositol phosphate accumulation, cAMP levels,intracellular calcium mobilization, and potassium currents, for example;to systems measuring these same second messengers but which have beenmodified or adapted to be of higher throughput, more generic and moresensitive; to cell based assays reporting more general cellular eventsresulting from receptor activation such as metabolic changes,differentiation, cell division/proliferation. Description of severalsuch assays follow.

Cyclic AMP (cAMP) assay

The receptor-mediated stimulation or inhibition of cyclic AMP (cAMP)formation may be assayed in cells expressing the receptors. Cells areplated in 96-well plates or other vessels and preincubated in a buffersuch as HEPES buffered saline (NaCl (150 mM), CaCl₂ (1 mM) , KCl (5 mM),glucose (10 mM)) supplemented with a phosphodiesterase inhibitor such as5 mM theophylline, with or without protease inhibitor cocktail (Forexample, a typical inhibitor cocktail contains 2 μg/ml aprotinin, 0.5mg/ml leupeptin, and 10 μg/ml phosphoramidon.) for 20 min at 37° C., in5% CO₂. Test compounds are added with or without 10 mM forskolin andincubated for an additional 10 min at 37° C. The medium is thenaspirated and the reaction stopped by the addition of 100 mM HCl orother methods. The plates are stored at 4° C. for 15 min, and the cAMPcontent in the stopping solution is measured by radioimmunoassay.Radioactivity may be quantified using a gamma counter equipped with datareduction software. Specific modifications may be performed to optimizethe assay for the receptor or to alter the detection method of cAMP.

Arachidonic acid release assay

Cells expressing the receptor are seeded into 96 well plates or othervessels and grown for 3 days in medium with supplements. ³H-arachidonicacid (specific activity =0.75 μCi/ml) is delivered as a 100 μL aliquotto each well and samples are incubated at 37° C., 5% CO₂ for 18 hours.The labeled cells are washed three times with medium. The wells are thenfilled with medium and the assay is initiated with the addition of testcompounds or buffer in a total volume of 250 μL. Cells are incubated for30 min at 37° C., 5% CO₂. Supernatants are transferred to a microtiterplate and evaporated to dryness at 75° C. in a vacuum oven. Samples arethen dissolved and resuspended in 25 μL distilled water. Scintillant(300 μL) is added to each well and samples are counted for ³H in aTrilux plate reader. Data are analyzed using nonlinear regression andstatistical techniques available in the GraphPAD Prism package (SanDiego, Calif.).

Inositol phosphate assay

SNORF25 receptor-mediated activation of the inositol phosphate (IP)second messenger pathways can be assessed by radiometric measurement ofIP products.

In a 96 well microplate format assay, cells are plated at a density of70,000 cells per well and allowed to incubate for 24 hours. The cellsare then labeled with 0.5 μCi [³H]-myo-inositol overnight at 37° C., 5%CO₂. Immediately before the assay, the medium is removed and replacedwith 90 μL of PBS containing 10 mM LiCl. The plates are then incubatedfor 15 min at 37° C., 5% CO₂. Following the incubation, the cells arechallenged with agonist (10 μl/well; 10× concentration) for 30 min at37° C., 5% CO₂. The challenge is terminated by the addition of 100 μL of50% v/v trichloroacetic acid, followed by incubation at 4° C. forgreater than 30 minutes. Total IPs are isolated from the lysate by ionexchange chromatography. Briefly, the lysed contents of the wells aretransferred to a Multiscreen HV filter plate (Millipore) containingDowex AG1-X8 (200-400 mesh, formate form). The filter plates areprepared adding 100 μL of Dowex AG1-X8 suspension (50% v/v, water:resin) to each well. The filter plates are placed on a vacuum manifoldto wash or elute the resin bed. Each well is first washed 2 times with200 μl of 5 mM myo-inositol. Total [³H]inositol phosphates are elutedwith 75 μl of 1.2M ammonium formate/0.1M formic acid solution into96-well plates. 200 μL of scintillation cocktail is added to each well,and the radioactivity is determined by liquid scintillation counting.

Intracellular calcium mobilization assays

The intracellular free calcium concentration may be measured bymicrospectrofluorimetry using the fluorescent indicator dye Fura-2/AM(Bush et al, 1991). Cells expressing the receptor are seeded onto a 35mm culture dish containing a glass coverslip insert and allowed toadhere overnight. Cells are then washed with HBS and loaded with 100 μLof Fura-2/AM (10 μM) for 20 to 40 min. After washing with HBS to removethe Fura-2/AM solution, cells are equilibrated in HBS for 10 to 20 min.Cells are then visualized under the 40× objective of a Leitz Fluovert FSmicroscope and fluorescence emission is determined at 510 nM withexcitation wavelengths alternating between 340 nM and 380 nM. Rawfluorescence data are converted to calcium concentrations using standardcalcium concentration curves and software analysis techniques.

In another method, the measurement of intracellular calcium can also beperformed on a 96-well (or higher) format and with alternativecalcium-sensitive indicators, preferred examples of these are: aequorin,Fluo-3, Fluo-4, Fluo-5, Calcium Green-1, Oregon Green, and 488 BAPTA.After activation of the receptors with agonist ligands the emissionelicited by the change of intracellular calcium concentration can bemeasured by a luminometer, or a fluorescence imager; a preferred exampleof this is the fluorescence imager plate reader (FLIPR).

Cells expressing the receptor of interest are plated into clear,flat-bottom, black-wall 96-well plates (Costar) at a density of80,000-150,000 cells per well and allowed to incubate for 48 hr at 5%CO₂, 37° C. The growth medium is aspirated and 100 μl of loading mediumcontaining fluo-3 dye is added to each well. The loading mediumcontains: Hank's BSS (without phenol red) (Gibco), 20 mM HEPES (Sigma),0.1 or 1% BSA (Sigma), dye/pluronic acid mixture (e.g. 1 mM Flou-3, AM(Molecular Probes) and 10% pluronic acid (Molecular Probes) mixedimmediately before use), and 2.5 mM probenecid (Sigma)(prepared fresh).The cells are allowed to incubate for about 1 hour at 5% CO₂, 37° C.

During the dye loading incubation the compound plate is prepared. Thecompounds are diluted in wash buffer (Hank's BSS (without phenol red),20 mM HEPES, 2.5 mM probenecid) to a 4× final concentration andaliquoted into a clear v-bottom plate (Nunc). Following the incubationthe cells are washed to remove the excess dye. A Denley plate washer isused to gently wash the cells 4 times and leave a 100 μl final volume ofwash buffer in each well. The cell plate is placed in the center trayand the compound plate is placed in the right tray of the FLIPR. TheFLIPR software is setup for the experiment, the experiment is run andthe data are collected. The data are then analyzed using an excelspreadsheet program.

Antagonist ligands are identified by the inhibition of the signalelicited by agonist ligands.

GTPγS functional assay

Membranes from cells expressing the receptor are suspended in assaybuffer (e.g., 50 mM Tris, 100 mM NaCl, 5 mM MgCl₂, 10 μM GDP, pH 7.4)with or without protease inhibitors (e.g., 0.1% bacitracin). Membranesare incubated on ice for 20 minutes, transferred to a 96-well Milliporemicrotiter GF/C filter plate and mixed with GTPγ³⁵S (e.g., 250,000cpm/sample, specific activity ˜1000 Ci/mmol) plus or minus unlabeledGTP_(Y)S (final concentration=100 μM). Final membrane proteinconcentration≈90 μg/ml. Samples are incubated in the presence or absenceof test compounds for 30 min. at room temperature, then filtered on aMillipore vacuum manifold and washed three times with cold (4° C.) assaybuffer. Samples collected in the filter plate are treated withscintillant and counted for ³⁵S in a Trilux (Wallac) liquidscintillation counter. It is expected that optimal results are obtainedwhen the receptor membrane preparation is derived from an appropriatelyengineered heterologous expression system, i.e., an expression systemresulting in high levels of expression of the receptor and/or expressingG-proteins having high turnover rates (for the exchange of GDP for GTP).GTPγS assays are well-known to those skilled in the art, and it iscontemplated that variations on the method described above, such as aredescribed by Tian et al. (1994) or Lazareno and Birdsall (1993), may beused.

Microphysiometric assay

Because cellular metabolism is intricately involved in a broad range ofcellular events (including receptor activation of multiple messengerpathways), the use of microphysiometric measurements of cell metabolismcan in principle provide a generic assay of cellular activity arisingfrom the activation of any receptor regardless of the specifics of thereceptor's signaling pathway.

General guidelines for transient receptor expression, cell preparationand microphysiometric recording are described elsewhere (Salon, J. A.and Owicki, J. A., 1996). Typically cells expressing receptors areharvested and seeded at 3×10⁵ cells per microphysiometer capsule incomplete media 24 hours prior to an experiment. The media is replacedwith serum free media 16 hours prior to recording to minimizenon-specific metabolic stimulation by assorted and ill-defined serumfactors. On the day of the experiment the cell capsules are transferredto the microphysiometer and allowed to equilibrate in recording media(low buffer RPMI 1640, no bicarbonate, no serum (Molecular DevicesCorporation, Sunnyvale, Calif.) containing 0.1% fatty acid free BSA),during which a baseline measurement of basal metabolic activity isestablished.

A standard recording protocol specifies a 100 μl/min flow rate, with a 2min total pump cycle which includes a 30 sec flow interruption duringwhich the acidification rate measurement is taken. Ligand challengesinvolve a 1 min 20 sec exposure to the sample just prior to the firstpost challenge rate measurement being taken, followed by two additionalpump cycles for a total of 5 min 20 sec sample exposure. Typically,drugs in a primary screen are presented to the cells at 10 μM finalconcentration. Follow up experiments to examine dose-dependency ofactive compounds are then done by sequentially challenging the cellswith a drug concentration range that exceeds the amount needed togenerate responses ranging from threshold to maximal levels. Ligandsamples are then washed out and the acidification rates reported areexpressed as a percentage increase of the peak response over thebaseline rate observed just prior to challenge.

MAP kinase assay

MAP kinase (mitogen activated kinase) may be monitored to evaluatereceptor activation. MAP kinase is activated by multiple pathways in thecell. A primary mode of activation involves the ras/raf/MEK/MAP kinasepathway. Growth factor (tyrosine kinase) receptors feed into thispathway via SHC/Grb-2/SOS/ras. Gi coupled receptors are also known toactivate ras and subsequently produce an activation of MAP kinase.Receptors that activate phospholipase C (such as Gq/G11-coupled) producediacylglycerol (DAG) as a consequence of phosphatidyl inositolhydrolysis. DAG activates protein kinase C which in turn phosphorylatesMAP kinase.

MAP kinase activation can be detected by several approaches. Oneapproach is based on an evaluation of the phosphorylation state, eitherunphosphorylated (inactive) or phosphorylated (active). Thephosphorylated protein has a slower mobility in SDS-PAGE and cantherefore be compared with the unstimulated protein using Westernblotting. Alternatively, antibodies specific for the phosphorylatedprotein are available (New England Biolabs) which can be used to detectan increase in the phosphorylated kinase. In either method, cells arestimulated with the test compound and then extracted with Laemmlibuffer. The soluble fraction is applied to an SDS-PAGE gel and proteinsare transferred electrophoretically to nitrocellulose or Immobilon.Immunoreactive bands are detected by standard Western blottingtechnique. Visible or chemiluminescent signals are recorded on film andmay be quantified by densitometry.

Another approach is based on evaluation of the MAP kinase activity via aphosphorylation assay. Cells are stimulated with the test compound and asoluble extract is prepared. The extract is incubated at 30° C. for 10min with gamma-³²P-ATP, an ATP regenerating system, and a specificsubstrate for MAP kinase such as phosphorylated heat and acid stableprotein regulated by insulin, or PHAS-I. The reaction is terminated bythe addition of H₃PO₄ and samples are transferred to ice. An aliquot isspotted onto Whatman P81 chromatography paper, which retains thephosphorylated protein. The chromatography paper is washed and countedfor ³²P in a liquid scintillation counter. Alternatively, the cellextract is incubated with gamma-³²P-ATP, an ATP regenerating system, andbiotinylated myelin basic protein bound by streptavidin to a filtersupport. The myelin basic protein is a substrate for activated MAPkinase. The phosphorylation reaction is carried out for 10 min at 30° C.The extract can then by aspirated through the filter, which retains thephosphorylated myelin basic protein. The filter is washed and countedfor ³²P by liquid scintillation counting.

Cell proliferation assay

Receptor activation of the receptor may lead to a mitogenic orproliferative response which can be monitored via ³H-thymidine uptake.When cultured cells are incubated with ³H-thymidine, the thymidinetranslocates into the nuclei where it is phosphorylated to thymidinetriphosphate. The nucleotide triphosphate is then incorporated into thecellular DNA at a rate that is proportional to the rate of cell growth.Typically, cells are grown in culture for 1-3 days. Cells are forcedinto quiescence by the removal of serum for 24 hrs. A mitogenic agent isthen added to the media. Twenty-four hours later, the cells areincubated with ³H-thymidine at specific activities ranging from 1 to 10μCi/ml for 2-6 hrs. Harvesting procedures may involve trypsinization andtrapping of cells by filtration over GF/C filters with or without aprior incubation in TCA to extract soluble thymidine. The filters areprocessed with scintillant and counted for ³H by liquid scintillationcounting. Alternatively, adherent cells are fixed in MeOH or TCA, washedin water, and solubilized in 0.05% deoxycholate/0.1N NaOH. The solubleextract is transferred to scintillation vials and counted for ³H byliquid scintillation counting.

Alternatively, cell proliferation can be assayed by measuring theexpression of an endogenous or heterologous gene product, expressed bythe cell line used to transfect the receptor, which can be detected bymethods such as, but not limited to, florescence intensity, enzymaticactivity, immunoreactivity, DNA hybridization, polymerase chainreaction, etc.

Promiscuous second messenger assays

It is not possible to predict, a priori and based solely upon the GPCRsequence, which of the cell's many different signaling pathways anygiven receptor will naturally use. It is possible, however, to coaxreceptors of different functional classes to signal through apre-selected pathway through the use of promiscuous G_(α)subunits. Forexample, by providing a cell based receptor assay system with anendogenously supplied promiscuous G_(l) subunit such as G_(α15) orG_(α16) or a chimeric G_(α)subunit such as G_(αqz), a GPCR, which mightnormally prefer to couple through a specific signaling pathway (e.g.,G_(s), G_(i), G_(q), G₀, etc.), can be made to couple through thepathway defined by the promiscuous G_(α)subunit and upon agonistactivation produce the second messenger associated with that subunit'spathway. In the case of G_(α15), G_(α16) and/or G_(αqz) this wouldinvolve activation of the G_(q) pathway and production of the secondmessenger IP₃. Through the use of similar strategies and tools, it ispossible to bias receptor signaling through pathways producing othersecond messengers such as Ca⁺⁺, cAMP, and K⁺ currents, for example(Milligan, 1999).

It follows that the promiscuous interaction of the exogenously suppliedG_(α)subunit with the receptor alleviates the need to carry out adifferent assay for each possible signaling pathway and increases thechances of detecting a functional signal upon receptor activation.

Methods for recording currents in Xenopus oocytes

Oocytes are harvested from Xenopus laevis and injected with mRNAtranscripts as previously described (Quick and Lester, 1994; Smith etal.,1997). The test receptor of this invention and Gα subunit RNAtranscripts are synthesized using the T7 polymerase (“Message Machine,”Ambion) from linearized plasmids or PCR products containing the completecoding region of the genes. Oocytes are injected with 10 ng syntheticreceptor RNA and incubated for 3-8 days at 17 degrees. Three to eighthours prior to recording, oocytes are injected with 500 pg promiscuousGα subunits mRNA in order to observe coupling to Ca⁺⁺ activated Cl⁻currents. Dual electrode voltage clamp (Axon Instruments Inc.) isperformed using 3M KCl-filled glass microelectrodes having resistancesof 1-2 MOhm. Unless otherwise specified, oocytes are voltage clamped ata holding potential of −80 mV. During recordings, oocytes are bathed incontinuously flowing (1-3 ml/min) medium containing 96 mM NaCl, 2 mMKCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES, pH 7.5 (ND96). Drugs areapplied either by local perfusion from a 10 μl glass capillary tubefixed at a distance of 0.5 mm from the oocyte, or by switching from aseries of gravity fed perfusion lines.

Other oocytes may be injected with a mixture of receptor mRNAs andsynthetic mRNA encoding the genes for G-protein-activated inwardrectifier channels (GIRK1 and GIRK4, U.S. Pat. Nos. 5,734,021 and5,728,535 or GIRK1 and GIRK2) or any other appropriate combinations(see, e.g., Inanobe et al., 1999). Genes encoding G-protein inwardlyrectifying K⁺ (GIRK) channels 1, 2 and 4 (GIRK1, GIRK2, and GIRK4) maybe obtained by PCR using the published sequences (Kubo et al., 1993;Dascal et al., 1993; Krapivinsky et al., 1995 and 1995b) to deriveappropriate 5′ and 3′ primers. Human heart or brain cDNA may be used astemplate together with appropriate primers.

Heterologous expression of GPCRs in Xenopus oocytes has been widely usedto determine the identity of signaling pathways activated by agoniststimulation (Gundersen et al., 1983; Takahashi et al., 1987). Activationof the phospholipase C (PLC) pathway is assayed by applying a testcompound in ND96 solution to oocytes previously injected with mRNA forthe SNORF25 receptor and observing inward currents at a holdingpotential of approximately −80 mV. The appearance of currents thatreverse at −25 mV and display other properties of the Ca⁺⁺-activated Cl⁻channel is indicative of receptor-activation of PLC and release of IP₃and intracellular Ca⁺⁺. Such activity is exhibited by GPCRs that coupleto G_(q) or G₁₁.

Involvement of the G_(i/o) class of G-proteins in GPCR-stimulatedCa⁺⁺-activated Cl⁻ currents is evaluated using PTX, a toxin whichinactivates G_(i/o) G-proteins. Oocytes are injected with 25 ngPTX/oocyte and modulation of Ca⁺⁺-activated Cl⁻ currents by SNORF25receptor is evaluated 2-5 h subsequently.

Elevation of intracellular cAMP can be monitored in oocytes byexpression of the cystic fibrosis transmembrane conductance regulator(CFTR) whose Cl⁻-selective pore opens in response to phosphorylation byprotein kinase A (Riordan, 1993). In order to prepare RNA transcriptsfor expression in oocytes, a template was created by PCR using 5′ and 3′primers derived from the published sequence of the CFTR gene (Riordan,1993). The 5′ primer included the sequence coding for T7 polymerase sothat transcripts could be generated directly from the PCR productswithout cloning. Oocytes were injected with 10 ng of CFTR mRNA inaddition to 10-15 ng mRNA for SNORF25. Electrophysiological recordingswere made in ND96 solution after a 2-3 day incubation at 18° C. Currentsare recorded under dual electrode voltage clamp (Axon Instruments Inc.)with 3M KCl-filled glass microelectrodes having resistances of 1-2 Mohm.Unless otherwise specified, oocytes are voltage clamped at a holdingpotential of −80 mV. During recordings, oocytes are bathed incontinuously flowing (1-3 ml/min) medium containing 96 mM NaCl, 2 mMKCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES, pH 7.5 (ND96). Drugs areapplied either by local perfusion from a 10 μl glass capillary tubefixed at a distance of 0.5 mm from the oocyte, or by switching from aseries of gravity fed perfusion lines.

Activation of G-protein G_(i) and G_(o) can be monitored by measuringthe activity of inwardly rectifying K⁺ (potassium) channels (GIRKs).Activity may be monitored in oocytes that have been co-injected withmRNAs encoding the mammalian receptor plus GIRK subunits. GIRK geneproducts co-assemble to form a G-protein activated potassium channelknown to be activated (i.e., stimulated) by a number of GPCRs thatcouple to G_(i) or G_(o) (Kubo et al., 1993; Dascal et al., 1993).Oocytes expressing the mammalian receptor plus the GIRK subunits aretested for test compound responsivity by measuring K⁺ currents inelevated K⁺ solution containing 49 mM K⁺.

Localization of mRNA coding for human and rat SNORF25.

Methods: Quantitative RT-PCR using a fluorogenic probe with real timedetection.

Quantitative RT-PCR using fluorogenic probes and a panel of mRNAextracted from human and rat tissue was used to characterize thelocalization of SNORF25 rat and human RNA.

This assay utilizes two oligonucleotides for conventional PCRamplification and a third specific oligonucleotide probe that is labeledwith a reporter at the 5′ end and a quencher at the 3′ end of theoligonucleotide. In the instant invention, FAM (6-carboxyfluorescein)and JOE (6 carboxy-4.5-dichloro-2,7-dimethoxyfluorescein) were the tworeporters that were utilized and TAMRA(6-carboxy-4,7,2,7′-tetramethylrhodamine) was the quencher. Asamplification progresses, the labelled oligonucleotide probe hybridizesto the gene sequence between the two oligonucleotides used foramplification. The nuclease activity of Taq, or rTth thermostable DNApolymerases is utilized to cleave the labelled probe. This separates thequencher from the reporter and generates a fluorescent signal that isdirectly proportional to the amount of amplicon generated. This labelledprobe confers a high degree of specificity. Non-specific amplificationis not detected as the labelled probe does not hybridize. Allexperiments were conducted in a PE7700 Sequence Detection System (PerkinElmer, Foster City, Calif.).

Quantitative RT-PCR

For the detection of RNA encoding SNORF25, quantitative RT-PCR wasperformed on mRNA extracted from tissue. Reverse transcription and PCRreactions were carried out in 50 μl volumes using rTth thermostable DNApolymerase (Perkin Elmer). Primers with the following sequences wereused:

SNORF 25 human: Forward primer: SNORF25H-765F5′-CCTCTACCTAGTGCTGGAACGG-3′ (SEQ ID NO: 18) Reverse primerSNORF25H-868R 5′-GCTGCAGTCGCACCTCCT-3′ (SEQ ID NO: 19) Fluorogenicoligonucleotide probe: SNORF25H-814T5′ (6-FAM)-TCCCTGCTCAACCCACTCATCTATGCCTATT-(TAMRA) 3′ (SEQ ID NO: 20)SNORF25 rat forward primer SNORF25R-231F 5′-GTGTAGCCTTCGGATGGCA-3′ (SEQID NO: 21) reverse primer SNORF25R-32 9R 5′-GGCTGCTTAATGGCCAGGTAC-3′(SEQ ID NO: 22) Fluorogenic oligonucleotide probe: SNORF2SR-278T5′ (6-FAM)-TCCTCACGGTCATGCTGATTGCCTTT-(TAMRA)3′ (SEQ ID NO: 23)

Using these primer pairs, amplicon length is 104 bp for human SNORF25and 99 bp for rat SNORF25. Each RT-PCR reaction contained 50 ng mRNA.Oligonuceotide concentrations were: 500 nM of forward and reverseprimers, and 200 nM of fluorogenic probe. Concentrations of reagents ineach reaction were: 300 μM each of dGTP; dATP; dCTP; 600 μM UTP; 3.0 mMMn(OAc)2; 50 mM Bicine; 115 mM potassium acetate, 8% glycerol, 5 unitsrTth thermostable DNA polymerase, and 0.5 units of uracil N-glycosylase.Buffer for RT-PCR reactions also contained a fluor used as a passivereference (ROX: Perkin Elmer proprietary passive reference I). Allreagents for RT-PCR (except mRNA and oligonucleotide primers) wereobtained from Perkin Elmer (Foster City, Calif.). Reactions were carriedusing the following thermal cycler profile: 50° C. 2 min., 60° C. 30min., 95° C. 5 min., followed by 40 cycles of: 94° C., 20 sec., 62° C. 1min.

Positive controls for PCR reactions consisted of amplification of thetarget sequence from a plasmid construct. Standard curves forquantitation were constructed using the human SNORF25 gene in a plasmidvector or RNA extracted from pancreas as a template for amplification.Negative controls consisted of mRNA blanks, as well as primer and mRNAblanks. To confirm that the mRNA was not contaminated with genomic DNA,PCR reactions were carried out without reverse transcription using TaqDNA polymerase. Integrity of RNA was assessed by amplification of mRNAcoding for cyclophilin or glyceraldehyde 3-phosphate dehydrogenase(GAPDH). Following reverse transcription and PCR amplification, data wasanalyzed using Perkin Elmer sequence detection software. The fluorescentsignal from each well was normalized using an internal passivereference, and data was fitted to a standard curve to obtain relativequantities of SNORF25 mRNA expression.

RESULTS AND DISCUSSION

Cloning of the full-length sequence of SNORF25

Genomic DNA and cDNA prepared from several tissues (including GH1 cellsand Rin14b cells) was subjected to MOPAC PCR with two degenerate primersdesigned based on the third transmembrane domain of the members of thegalanin, somatostatin, and opioid receptor families and the seventhtransmembrane domain of members of the galanin receptor family. Threeproducts from this reaction were found to be the same clone in eitherorientation (forward or reverse), which was a novel sequence not foundin the Genbank, SwissProtPlus, GSS, EST, or STS databases. It containedsignificant homology to other known G protein-coupled receptors (˜29%identity to the known receptors dopamine D1, beta-adrenergic 2b and5-HT_(1F); 34% identity to 5-HT_(4L) receptor). This receptor sequencewas later named SNORF25, and was used to design primers for 5′ and 3′Rapid Amplification of cDNA Ends (RACE), as described in the Methodssection above. The 5′ RACE reaction yielded sequence information throughthe first transmembrane domain and a putative in-frame initiatingmethionine-coding sequence surrounded by a kozak consensus sequence(ACCATGG).

The 3′ RACE reaction yielded a 600 bp band by agarose gelelectrophoresis. This band was subcloned into the TA cloning kit, andisolated colonies were sequenced. The sequence of these productsrevealed the presence of an in-frame stop codon downstream from theregion coding for the seventh transmembrane domain. The entire size ofthe coding sequence of SNORF25 was determined to be 1005 bp, coding fora protein of 335 amino acids. Two primers, JAB86 and JAB84, were used toamplify the entire coding sequence from Rin14b cell line cDNA and ratgenomic DNA using the Expand Long PCR system. The primers for thisreaction were specific to the 5′ and 3′ untranslated regions of SNORF25with BamHI and HindIII restriction sites incorporated into the 5′ endsof the 5′ and 3′ primers, respectively. When the products of thesereactions were subcloned into pcDNA3.1(−) and sequenced, the sequence ofthe Rin14b clone and the genomic clone were found to be identical, andthe vector construct containing rat SNORF25 was named pcDNA3.1-rSNORF25.

Hydophobicity (Kyte-Doolittle) analysis of the amino acid sequence ofthe full-length clone indicates the presence of seven hydrophobicregions, which is consistent with the seven transmembrane domains of a Gprotein-coupled receptor. The seven expected transmembrane domains areindicated in FIG. 4. A comparison of nucleotide and peptide sequences ofrat SNORF25 with sequences contained in the Genbank, EMBL, andSwissProtPlus databases reveals that the amino acid sequence of thisreceptor is most related to histamine, adenosine, serotonin, betaadrenergic, and dopamine receptor families, displaying between 25-30%overall amino acid identity with these receptors. The N- and C-terminiare relatively short, much like the adenosine receptor family. However,transmembrane domain analysis indicates that this receptor shares asignificant degree of identity to other GPCRs in its transmembranedomains. A comparison of all of the transmembrane domains of SNORF25simultaneously with a comprehensive list of GPCR transmembrane domainswould suggest that the transmembrane domains of SNORF25 have the highestdegree of identity with the beta adrenergic receptors 1 and 2 of 31% and32%, respectively, as well as 5-HT₇ and 5-hT_(5B) receptors of 32% and36.6%, respectively. When transmembrane domains are analyzedindividually by a FASTA search, SNORF25 exhibits considerable similarityto the transmembrane domains of a variety of known G protein-coupledreceptors.

In order to clone the human homolog of SNORF25, a human genomic cosmidlibrary was screened at medium stringency with labelled oligonucleotideprobes designed based on the second and fifth transmembrane domains ofrat SNORF25. Out of roughly 225,000 colonies screened, two colonieshybridized to the probes. After isolation and analysis of each colony,these two clones were determined to be identical cosmid clonescontaining the human homolog of SNORF25. Southern blot analysis ofseveral restriction digests of this cosmid and subsequent sequencing ofpositive bands indicated that a BamHI/HindIII digest of this cosmidyielded a 1.9 kb fragment containing the full-length coding sequence ofthis human clone. The construct of the human receptor subcloned into theBamHI/HindIII site of the pEXJT3T7 vector is named pEXJT3T7-hSNORF25.Human SNORF25 exhibits an 80% DNA identity and 83% amino acid identityto rat SNORF25. Like the rat receptor, the protein-coding region ofhuman SNORF25 is 1005 nucleotides (FIGS. 1A-1B), coding for a protein of335 amino acids (FIGS. 2A-2B). The DNA and amino acid sequences of ratSNORF25 are shown in FIGS. 3A-3B and 4A-4B, respectively.

A search of the GenEMBL, SwissProtPlus, EST, STS and GSS databasesconfirmed that human SNORF25 is also a unique novel sequence. Other thanits identity with rat SNORF25, it shares 28-30% overall identity withadenosine 2a, 5-HT_(4L), 5-HT_(4S), 5-H_(T6), and 5-HT₇, dopamine D₁ andD₅, and somatostatin 5 receptors. It also shares 25-26% identity withadenosine A1, histamine H1 and 2, beta adrenergic 1, and somatostatin 2and 3 receptors. A comparison of all of the transmembrane domains ofhuman SNORF25 simultaneously with a comprehensive list of GPCRtransmembrane domains would suggest that the transmembrane domains ofhuman SNORF25 have the highest degree of identity with the beta 1 and 2adrenergic receptors (29% and 32%, respectively) and 5-HT₄. Individualtransmembrane domains of human SNORF25 share significant identity withtransmembrane domains from several other G protein-coupled receptors.

Both rat and human SNORF25 have several potential protein kinase C (PKC)phosphorylation motifs throughout their amino acid sequences. For bothreceptors, threonine 73, serine 79, and serine 309 are potential PKCphosphorylation sites. The human receptor has an additional putative PKCphosphorylation site at serine 214, which is a proline in rat SNORF25.Both receptors share a potential casein kinase II (CKII) phosphorylationsite at serine 329. The human SNORF25 also contains two more potentialCKII phosphorylation sites, threonine 217 and serine 331, that are notpresent in the rat receptor. Conversely, rat SNORF25 contains apotential tyrosine phosphorylation site at tyrosine 323, which is notpresent in the human receptor.

cAMP response of SNORF25-transfected cells

The expression vector (pcDNA) containing the SNORF25 cDNA wastransfected by electroporation method into CHO cells. After plating, thetransfectants were challenged with a ligand library that included, amongother things, several of the traditional neurotransmitters such ashistamine, adenosine, serotonin, norepinephrine, and dopamine, based onhomology of SNORF25 to the receptors of these ligands (see above), andtested for their ability to stimulate cAMP or IP release abovemock-transfected cells. Interestingly, the basal cAMP levels ofSNORF25-transfected cells were significantly higher (>10-fold) thanmock-transfected cells (FIG. 5). This observation suggested that SNORF25receptor may functionally be coupled to a cAMP stimulatory pathway.Among the ligands tested, only all-trans retinoic acid (ATRA) produced asignificant increase in cAMP but not IP release in SNORF25-transfectedcells, without affecting these parameters in mock-transfected CHO cells.The response produced at 10 μM concentration of ATRA (2- to 5-fold abovebasal) was comparable to that produced by forskolin, a potent directstimulator of adenylyl cyclase (FIG. 6) (n=3).

Responses to forskolin in both mock- and SNORF25-transfected Cos-7 cellswere almost identical (FIG. 6), suggesting that the enhanced maximalresponse to ATRA observed in SNORF25-expressing cells, as compared tomock DNA-transfected cells, was not due to a change in cell density orin the intrinsic properties of the cells. All-trans retinol (vitaminA₁), a close analogue of ATRA failed to produce an increase in cAMP at10 μM (FIG. 6).

Subsequent experiments demonstrated that the ATRA-induced increase incAMP formation was independent of host cell as it was observed also inCos-7 cells (n=3) (FIG. 7). All-trans-retinoic acid produced no responsein Cos-7 cells transfected with other known cyclase-stimulatoryreceptors including dopamine D1, D5, serotonin 5-HT4 and 5-HT6receptors, indicating that the response observed to ATRA is specific toSNORF25-transfected cells (FIG. 7).

The cAMP response to ATRA in Cos-7 cells was concentration-dependentwith EC₅₀ values ranging from approximately 0.2 to 1 μM and E_(max) ofapproximately 200-300% (FIG. 8).

Activation of calcium-activated Cl⁻ currents in SNORF25 expressingXenopus oocytes

Elevation of intracellular cAMP can be monitored in oocytes byexpression of the cystic fibrosis transmembrane conductance regulator(CFTR) whose Cl⁻-selective pore opens in response to phosphorylation byprotein kinase A (Riordan, 1993). The activity of SNORF25 was thereforetested in oocytes co-injected with mRNA encoding SNORF25 and mRNAencoding CFTR. In 17 out of 39 of these ooctyes an inward Cl⁻ current(105±20 nA) was measured in response to the application of 10 μMall-trans-retinoic acid (See FIGS. 9A-9C and 10).

This response was specific to the expression of SNORF25 since no suchcurrent was observed in other oocytes injected with only mRNA encodingthe CFTR channel. Similar currents were observed in oocytes injectedwith the β2-adrenergic receptor (B2AR) (See FIG. 9C), although thecurrents generated by SNORF25-expressing oocytes were generally 2-3 foldslower and smaller. All-trans-retinoic acid did not stimulate Cl⁻currents in oocytes lacking CFTR, indicating that the Gq-mediatedphospholipase C pathway was not activated. Responses also were notevoked in ooctyes expressing chimeric G-proteins which are able tocouple Gi and Go coupled GPCRs to the phospholipase C pathway. Takentogether, these observations support the hypothesis that SNORF25 encodesa GPCR which binds all-trans-retinoic acid and stimulates the productionof cAMP, presumably via activation of Gs.

In other systems, all-trans-retinoic acid stimulates one of severalnuclear receptors (see background) . This results in the enhancement oftranscription of one or more genes. SNORF25 expression in oocytes couldresult in the expression of a nuclear receptor for all-trans-retinoicacid, not normally present in uninjected oocytes, that when stimulatedproduces an elevation of cAMP. If this were the case, then retinoic acidwould not necessarily bind the SNORF25 receptor, but would act on apreviously know or novel nuclear receptor for retinoic acid. Thisindirect mechanism of action of retinoic acid may explain why the ligandfailed to elicit a CFTR response in 3 out of 6 batches of oocytes (17 of39 oocytes), and why the kinetics of CFTR activation were 2-3 timesslower than those observed under conditions where responses were evokedby activation of well-characterized GPCRs such as the B2 adrenergicreceptor (FIG. 9C). Nevertheless, the delay for activation of CFTR byretinoic acid was on the order of 10 seconds, and the activation ofnuclear receptors is typically in the range of several minutes to hours.Thus, while we cannot rule out an indirect mechanism of action ofretinoic acid, the relatively rapid onset of the response inSNORF25-expressing oocytes suggests that such a mechanism is unlikely.

Detection of mRNA coding for human SNORF25:

mRNA was isolated from multiple tissues (listed in Table 1) and assayedas described.

Quantitative RT-PCR using a fluorgenic probe demonstrated expression ofmRNA encoding human SNORF25 in most tissues assayed (Table 1). Highestlevels of human SNORF25 mRNA are found in the pancreas, stomach, smallintestine and fetal liver, with lower levels detected elsewhere. Mostnervous system structures showed little expression of SNORF25 mRNA ascompared to peripheral organs.

The highest levels of SNORF25 expression are found in the pancreas. Thepancreas secretes a variety of broadly active substances (includinginsulin), indicating that SNORF25 may play a role in regulating multiplemetabolic functions, potentially via endocrine mechanisms. SNORF25expression in the pancreas is not surprising as SNORF25 is alsoexpressed in a rat insulinoma cell line. This finding as well as thedetection of SNORF25 mRNA in liver indicate a possible role in theregulation of glucose levels and possibly diabetes.

Other organs with high levels of SNORF25 mRNA are stomach and smallintestine. The distribution to these structures is consistent withfunctions relating to gastrointestinal motility or absorption. It is notknown at this time if SNORF25 mRNA is localized to smooth muscle or tomucosal/submucosal layers.

Although detected in very low levels, the presence of SNORF25 mRNA inmultiple regions of the CNS including the thalamus and hippocampalformation (where levels are highest in the CNS) and other functionallydiverse areas, indicate a diffuse regulatory function or regionalfunctionality for this receptor.

Human SNORF25 mRNA appears to be developmentally regulated. In fetalliver, levels of mRNA approach those measured in adult pancreas (83%).However in adult tissue, this drops to less than 1% of the amount foundin the pancreas. The profound change of SNORF25 mRNA during developmentimplies a role in the maturation of the liver, or a role in theregulation of glucose demands/levels during development. The time courseof this increase has not been examined and would be important inunderstanding the function of this receptor.

In summary, the distribution of SNORF25 receptor mRNA implies broadregulatory functions that involves multiple organ systems, endocrinemechanisms, as well as the central nervous system.

Detection of mRNA coding for rat SNORF25

Unlike the restricted distribution of human SNORF25 mRNA, thedistribution of SNORF25 mRNA in the rat is widespread. One strikingdifference in the distribution between rat and human is the high levelsof SNORF25 mRNA detected in the rat central nervous system. In thehuman, the highest concentrations of SNORF25 mRNA are found in thepancreas, with very low levels found in CNS structures. In the rat thehighest levels of SNORF25 mRNA are found in the hippocampal formation,closely followed by levels detected in the cerebral cortex, cerebellum,hypothalamus, choroid plexus and medulla. SNORF25 mRNA is also detectedin both dorsal root and trigeminal ganglia. Although SNORF25 mRNA isdetected in rat pancreas and other peripheral organs, it is presentthere in much lower levels than in the CNS.

Rat SNORF25 was detected in most tissues assayed. In addition to thepancreas it is expressed in appreciable amounts in lung, colon,duodenum, ovary, kidney and the adrenal glands. It was detected in othertissues in decreasing amounts as shown in Table 2.

In summary, the broad distribution of rat SNORF25 receptor mRNA impliesbroad regulatory functions that involve multiple organ systems,endocrine mechanisms as well as the central nervous system. Thedifference in the distribution pattern seen between human and ratsuggests a broader, and potentially different role for this receptor inthe rat as compared to human.

TABLE 1 Distribution of mRNA coding for human SNORF25 receptors usinqqRT-PCR mRNA encoding SNORF25h is expressed as % of highest expressingtissue. qRT-PCR Region % of max Potential applications heart 0.31cardiovascular indications kidney 0.62 hypertension, electrolyte balanceliver 0.18 diabetes lung 0.32 respiratory disorders, asthma pancreas 100diabetes, endocrine disorders pituitary 0.03 endocrine/neuroendocrineregulation placenta 0.42 gestational abnormalities small intestine 4.63gastrointestinal disorders spleen 1.50 immune disorders stomach 12.60gastrointestinal disorders striated muscle 0.32 musculoskeletaldisorders amygdala 0.18 depression, phobias, anxiety, mood disorderscaudate-putamen 0.17 modulation of dopaminergic function cerebellum 0.06motor coordination cerebral cortex 0.01 sensory and motor integration,cognition hippocampus 0.27 cognition/memory spinal cord 0.00 analgesia,sensory modulation and transmission substantia nigra 0.05 modulation ofdopaminergic function. modulation of motor coordination. thalamus 0.60sensory integration fetal brain 0.14 developmental disorders fetal lung0.04 developmental disorders fetal kidney 0.90 developmental disordersfetal liver 82.63 developmental disorders

TABLE 2 Distribution of mRNA coding for rat SNORF25 receptors usingqRT-PCR mRNA encoding SNORF25r is expressed as % of highest expressingtissue. qRT-PCR Tissue % of max Potential applications adipose tissue9.08 metabolic disorders adrenal cortex 8.78 regulation of steroidhormones adrenal medulla 16.34 regulation of epinephrine release colon24.15 gastrointestinal disorders duodenum 18.89 gastrointestinaldisorders heart 11.98 cardiovascular indications kidney 15.86electrolyte balance, hypertension liver trace diabetes lung 32.57respiratory disorders, asthma ovary 17.74 reproductive function pancreas30.45 diabetes, endocrine disorders spleen not immune disorders detectedstomach 3.44 gastrointestinal disorders striated muscle 1.04musculoskeletal disorders testes 5.10 reproductive function urinarybladder 7.87 urinary incontinence vas deferens 7.16 reproductivefunction celiac plexus 17.82 modulation of autonomic innervationcerebellum 84.14 motor coordination cerebral cortex 83.54 Sensory andmotor integration, cognition choroid plexus 66.59 regulation ofcerebrospinal fluid dorsal root ganglia 38.14 sensory transmissionhippocampus 100 cognition/memory hypothalamus 67.19 appetite/obesityneuroendocrine regulation medulla 52.66 analgesia, motor coordinationolfactory bulb 6.66 olfaction pineal gland 41.16 regulation of melatoninrelease spinal cord 31.72 analgesia, sensory modulation and transmissiontrigeminal ganglia 42.98 sensory transmission

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23 1 1129 DNA Homo sapiens 1 tgagaatttc agctggagag atagcatgcc ctggtaagtgaagtcctgcc acttcgagac 60 atggaatcat ctttctcatt tggagtgatc cttgctgtcctggcctccct catcattgct 120 actaacacac tagtggctgt ggctgtgctg ctgttgatccacaagaatga tggtgtcagt 180 ctctgcttca ccttgaatct ggctgtggct gacaccttgattggtgtggc catctctggc 240 ctactcacag accagctctc cagcccttct cggcccacacagaagaccct gtgcagcctg 300 cggatggcat ttgtcacttc ctccgcagct gcctctgtcctcacggtcat gctgatcacc 360 tttgacaggt accttgccat caagcagccc ttccgctacttgaagatcat gagtgggttc 420 gtggccgggg cctgcattgc cgggctgtgg ttagtgtcttacctcattgg cttcctccca 480 ctcggaatcc ccatgttcca gcagactgcc tacaaagggcagtgcagctt ctttgctgta 540 tttcaccctc acttcgtgct gaccctctcc tgcgttggcttcttcccagc catgctcctc 600 tttgtcttct tctactgcga catgctcaag attgcctccatgcacagcca gcagattcga 660 aagatggaac atgcaggagc catggctgga ggttatcgatccccacggac tcccagcgac 720 ttcaaagctc tccgtactgt gtctgttctc attgggagctttgctctatc ctggaccccc 780 ttccttatca ctggcattgt gcaggtggcc tgccaggagtgtcacctcta cctagtgctg 840 gaacggtacc tgtggctgct cggcgtgggc aactccctgctcaacccact catctatgcc 900 tattggcaga aggaggtgcg actgcagctc taccacatggccctaggagt gaagaaggtg 960 ctcacctcat tcctcctctt tctctcggcc aggaattgtggcccagagag gcccagggaa 1020 agttcctgtc acatcgtcac tatctccagc tcagagtttgatggctaaga cggtaagggc 1080 agagaagttt caaagtgcct ttctcctccc actctggagccccaactag 1129 2 335 PRT Homo sapiens 2 Met Glu Ser Ser Phe Ser Phe GlyVal Ile Leu Ala Val Leu Ala Ser 1 5 10 15 Leu Ile Ile Ala Thr Asn ThrLeu Val Ala Val Ala Val Leu Leu Leu 20 25 30 Ile His Lys Asn Asp Gly ValSer Leu Cys Phe Thr Leu Asn Leu Ala 35 40 45 Val Ala Asp Thr Leu Ile GlyVal Ala Ile Ser Gly Leu Leu Thr Asp 50 55 60 Gln Leu Ser Ser Pro Ser ArgPro Thr Gln Lys Thr Leu Cys Ser Leu 65 70 75 80 Arg Met Ala Phe Val ThrSer Ser Ala Ala Ala Ser Val Leu Thr Val 85 90 95 Met Leu Ile Thr Phe AspArg Tyr Leu Ala Ile Lys Gln Pro Phe Arg 100 105 110 Tyr Leu Lys Ile MetSer Gly Phe Val Ala Gly Ala Cys Ile Ala Gly 115 120 125 Leu Trp Leu ValSer Tyr Leu Ile Gly Phe Leu Pro Leu Gly Ile Pro 130 135 140 Met Phe GlnGln Thr Ala Tyr Lys Gly Gln Cys Ser Phe Phe Ala Val 145 150 155 160 PheHis Pro His Phe Val Leu Thr Leu Ser Cys Val Gly Phe Phe Pro 165 170 175Ala Met Leu Leu Phe Val Phe Phe Tyr Cys Asp Met Leu Lys Ile Ala 180 185190 Ser Met His Ser Gln Gln Ile Arg Lys Met Glu His Ala Gly Ala Met 195200 205 Ala Gly Gly Tyr Arg Ser Pro Arg Thr Pro Ser Asp Phe Lys Ala Leu210 215 220 Arg Thr Val Ser Val Leu Ile Gly Ser Phe Ala Leu Ser Trp ThrPro 225 230 235 240 Phe Leu Ile Thr Gly Ile Val Gln Val Ala Cys Gln GluCys His Leu 245 250 255 Tyr Leu Val Leu Glu Arg Tyr Leu Trp Leu Leu GlyVal Gly Asn Ser 260 265 270 Leu Leu Asn Pro Leu Ile Tyr Ala Tyr Trp GlnLys Glu Val Arg Leu 275 280 285 Gln Leu Tyr His Met Ala Leu Gly Val LysLys Val Leu Thr Ser Phe 290 295 300 Leu Leu Phe Leu Ser Ala Arg Asn CysGly Pro Glu Arg Pro Arg Glu 305 310 315 320 Ser Ser Cys His Ile Val ThrIle Ser Ser Ser Glu Phe Asp Gly 325 330 335 3 1082 DNA Rattus norvegicus3 tcaagaccca gcatgccctt ataagtggga gtcctgctac ctcgaaccat ggagtcatct 60ttctcatttg gagtgatcct tgctgtcctg accatcctta tcattgctgt taatgcgctg 120gtggttgtgg ctatgctgct atcaatctac aagaatgatg gtgttggcct ttgcttcacc 180ttaaatctgg ccgtggctga taccttgatt ggcgtggcta tttctgggct agttacagac 240cagctctcca gctctgctca gcacacacag aagaccttgt gtagccttcg gatggcattc 300gtcacttctt ctgcagccgc ctctgtcctc acggtcatgc tgattgcctt tgacaggtac 360ctggccatta agcagcccct ccgttacttc cagatcatga atgggcttgt agccggagga 420tgcattgcag ggctgtggtt gatatcttac cttatcggct tcctcccact tggagtctcc 480atattccagc agaccaccta ccatgggccc tgcaccttct ttgctgtgtt tcacccaagg 540tttgtgctga ccctctcctg tgctggcttc ttcccagctg tgctcctctt tgtcttcttc 600tactgtgaca tgctcaagat tgcctctgtg cacagccagc acatccggaa gatggaacat 660gcaggagcca tggttggagc ttgccggccc ccacggcctg tcaatgactt caaggctgtc 720cggactgtat ctgtccttat tgggagcttc accctgtcct ggtctccgtt tctcatcact 780agcattgtgc aggtggcctg ccacaaatgc tgcctctacc aagtgctgga aaaatacctc 840tggctccttg gagttggcaa ctccctgctc aacccactca tctatgccta ttggcagagg 900gaggttcggc agcagctctg ccacatggcc ctgggggtga agaagttctt tacttcaatc 960ttcctccttc tctcggccag gaatcgtggt ccacagagga cccgagaaag ctcctatcac 1020atcgtcacta tcagccagcc ggagctcgat ggctaggatg gtaaggaatg gatgtttcca 1080ag 1082 4 335 PRT Rattus norvegicus 4 Met Glu Ser Ser Phe Ser Phe GlyVal Ile Leu Ala Val Leu Thr Ile 1 5 10 15 Leu Ile Ile Ala Val Asn AlaLeu Val Val Val Ala Met Leu Leu Ser 20 25 30 Ile Tyr Lys Asn Asp Gly ValGly Leu Cys Phe Thr Leu Asn Leu Ala 35 40 45 Val Ala Asp Thr Leu Ile GlyVal Ala Ile Ser Gly Leu Val Thr Asp 50 55 60 Gln Leu Ser Ser Ser Ala GlnHis Thr Gln Lys Thr Leu Cys Ser Leu 65 70 75 80 Arg Met Ala Phe Val ThrSer Ser Ala Ala Ala Ser Val Leu Thr Val 85 90 95 Met Leu Ile Ala Phe AspArg Tyr Leu Ala Ile Lys Gln Pro Leu Arg 100 105 110 Tyr Phe Gln Ile MetAsn Gly Leu Val Ala Gly Gly Cys Ile Ala Gly 115 120 125 Leu Trp Leu IleSer Tyr Leu Ile Gly Phe Leu Pro Leu Gly Val Ser 130 135 140 Ile Phe GlnGln Thr Thr Tyr His Gly Pro Cys Thr Phe Phe Ala Val 145 150 155 160 PheHis Pro Arg Phe Val Leu Thr Leu Ser Cys Ala Gly Phe Phe Pro 165 170 175Ala Val Leu Leu Phe Val Phe Phe Tyr Cys Asp Met Leu Lys Ile Ala 180 185190 Ser Val His Ser Gln His Ile Arg Lys Met Glu His Ala Gly Ala Met 195200 205 Val Gly Ala Cys Arg Pro Pro Arg Pro Val Asn Asp Phe Lys Ala Val210 215 220 Arg Thr Val Ser Val Leu Ile Gly Ser Phe Thr Leu Ser Trp SerPro 225 230 235 240 Phe Leu Ile Thr Ser Ile Val Gln Val Ala Cys His LysCys Cys Leu 245 250 255 Tyr Gln Val Leu Glu Lys Tyr Leu Trp Leu Leu GlyVal Gly Asn Ser 260 265 270 Leu Leu Asn Pro Leu Ile Tyr Ala Tyr Trp GlnArg Glu Val Arg Gln 275 280 285 Gln Leu Cys His Met Ala Leu Gly Val LysLys Phe Phe Thr Ser Ile 290 295 300 Phe Leu Leu Leu Ser Ala Arg Asn ArgGly Pro Gln Arg Thr Arg Glu 305 310 315 320 Ser Ser Tyr His Ile Val ThrIle Ser Gln Pro Glu Leu Asp Gly 325 330 335 5 21 DNA Artificial Sequencen = inosine 5 tbdsyvynga ymgntayvtk g 21 6 26 DNA Artificial Sequence n= inosine 6 ganrsnarng mrtanaynak nggrtt 26 7 25 DNA Artificial SequenceDescription of Artificial Sequence primer/probe 7 ttatgcttcc ggctcgtatgttgtg 25 8 27 DNA Artificial Sequence Description of Artificial Sequenceprimer/probe 8 atgtgctgca aggcgattta agttggg 27 9 20 DNA ArtificialSequence Description of Artificial Sequence primer/probe 9 tggtctgctggaatatggag 20 10 25 DNA Artificial Sequence Description of ArtificialSequence primer/probe 10 cttgggtgaa acacagcaaa gaagg 25 11 26 DNAArtificial Sequence Description of Artificial Sequence primer/probe 11atggaacatg caggagccat ggttgg 26 12 24 DNA Artificial SequenceDescription of Artificial Sequence primer/probe 12 aagacaaaga ggagcacagctggg 24 13 24 DNA Artificial Sequence Description of Artificial Sequenceprimer/probe 13 gctcaagatt gcctctgtgc acag 24 14 35 DNA ArtificialSequence Description of Artificial Sequence primer/probe 14 atctataagcttaggcactt ggaaacatcc attcc 35 15 36 DNA Artificial Sequence Descriptionof Artificial Sequence primer/probe 15 atctatggat cctgtgagaa tctgagctcaagaccc 36 16 58 DNA Artificial Sequence Description of ArtificialSequence primer/probe 16 ttcaccttaa atctggccgt ggctgatacc ttgattggcgtggctatttc tgggctag 58 17 61 DNA Artificial Sequence Description ofArtificial Sequence primer/probe 17 gctgtgtttc acccaaggtt tgtgctgaccctctcctgtg ctggcttctt cccagctgtg 60 c 61 18 22 DNA Artificial SequenceDescription of Artificial Sequence primer/probe 18 cctctaccta gtgctggaacgg 22 19 18 DNA Artificial Sequence Description of Artificial Sequenceprimer/probe 19 gctgcagtcg cacctcct 18 20 31 DNA Artificial SequenceDescription of Artificial Sequence primer/probe 20 tccctgctca acccactcatctatgcctat t 31 21 19 DNA Artificial Sequence Description of ArtificialSequence primer/probe 21 gtgtagcctt cggatggca 19 22 21 DNA ArtificialSequence Description of Artificial Sequence primer/probe 22 ggctgcttaatggccaggta c 21 23 26 DNA Artificial Sequence Description of ArtificialSequence primer/probe 23 tcctcacggt catgctgatt gccttt 26

What is claimed is:
 1. An isolated nucleic acid encoding a human or ratSNORF25 receptor, wherein the human SNORF25 receptor has an amino acidsequence identical to the amino acid sequence shown in FIGS. 2A-2B (SEQID NO: 2) or that encoded by plasmid pEXJT3T7-hSNORF25 (ATCC AccessionNo. 203495); and the rat SNORF25 receptor has an amino acid sequenceidentical to the amino acid sequence shown in FIGS. 4A-4B (SEQ ID NO: 4)or that encoded by plasmid pcDNA3.1-rSNORF25 (ATCC Accession No.203494).
 2. The nucleic acid of claim 1, wherein the nucleic acid isDNA.
 3. The DNA of claim 2, wherein the DNA is cDNA.
 4. The DNA of claim2, wherein the DNA is genomic DNA.
 5. The nucleic acid of claim 1,wherein the nucleic acid is RNA.
 6. A vector comprising the nucleic acidof claim
 1. 7. A vector of claim 6 adapted for expression in a cellwhich vector comprises the regulatory elements necessary for expressionof the nucleic acid in the cell operatively linked to the nucleic acidencoding the receptor so as to permit expression thereof, wherein thecell is a bacterial, amphibian, yeast, insect or mammalian cell.
 8. Thevector of claim 7, wherein the vector is a baculovirus.
 9. The vector ofclaim 6, wherein the vector is a plasmid.
 10. The plasmid of claim 9designated pEXJT3T7-hSNORF25 (ATCC Accession No. 203495).
 11. Theplasmid of claim 9 designated pcDNA3.1-rSNORF25 (ATCC Accession No.203494).
 12. A cell comprising the vector of claim
 9. 13. A cell ofclaim 12, wherein the cell is a non-mammalian cell.
 14. A cell of claim13, wherein the non-mammalian cell is a Xenopus oocyte cell or a Xenopusmelanophore cell.
 15. A cell of claim 12, wherein the cell is amammalian cell.
 16. A mammalian cell of claim 15, wherein the cell is aCOS-7 cell, a 293 human embryonic kidney cell, a NIH-3T3 cell, a LM(tk−)cell, a mouse Y1 cell, or a CHO cell.
 17. A cell of claim 12, whereinthe cell is an insect cell.
 18. An insect cell of claim 17, wherein theinsect cell is an Sf9 cell, an Sf21 cell or a Trichoplusia ni 5B-4 cell.19. A membrane preparation isolated from the cell of any one of claims12, 13, 15, 16, 17 or 18, wherein the membrane preparation comprisesrecombinantly produced SNORF25.
 20. A recombinant nucleic acidcomprising a nucleic acid encoding a human SNORF25 receptor, wherein thehuman SNORF25 receptor comprises an amino acid sequence identical to thesequence of the human SNORF25 receptor encoded by the nucleotidesequence beginning at the start codon at positions 61-63 and ending atthe stop codon at positions 1066-1068 as indicated in FIGS. 1A-1B (SEQID NO: 1).
 21. A recombinant nucleic acid comprising a nucleic acidencoding a rat SNORF25 receptor, wherein the rat SNORF25 receptorcomprises an amino acid sequence identical to the sequence of the ratSNORF25 receptor encoded by the nucleotide sequence beginning at thestart codon at positions 49-51 and ending at the stop codon at positions1054-1056 as indicated in FIGS. 3A-3B (SEQ ID NO: 3).